JP2013500256A - Oligomer-opioid agonist conjugates - Google Patents

Abstract

The present invention provides conjugates in which an opioid agonist, hydroxycodone (oxycodone), and hydrocodone (dihydrocodei π-one) are covalently bound to a poly (ethylene glycol) oligomer. The conjugates of the invention exhibit characteristics different from those of opioid agonists that are not conjugated to PEG oligomers when administered by any number of administration routes. The chemically modified opioid agonists of the present application relate to and / or have application (among others) in the fields of drug discovery, drug therapy, physiology, organic chemistry, and polymer chemistry.
[Selection figure] None

Description

(Cross-reference of related applications)
This application is based on US non-provisional patent application No. 12 / 558,395 filed on September 11, 2009, US provisional patent application No. 61 / 350,853, 2009 filed on June 2, 2010. Claims the benefit of priority over US provisional patent application 61 / 227,399, filed on July 21, 2009, the entire disclosures of which are hereby incorporated by reference. Incorporated in the description.

(Field)
The present invention provides chemically modified opioid agonists that have certain advantages over (among other things) opioid agonists that lack chemical modification. The chemically modified opioid agonists described herein relate to and / or have applications (among others) in the fields of drug discovery, drug therapy, physiology, organic chemistry, and macromolecular chemistry. .

  Opioid agonists such as morphine have been used for many years to treat patients suffering from pain. Opioid agonists exert analgesic and other pharmacological effects through interaction with opioid receptors, and there are three main types of opioid receptors: mu (μ) receptors and kappa (κ) receptors. There is a body, and a delta (δ) receptor. Opioid agonists typically have agonist activity at other opioid receptors (especially at increased concentrations), but most of the clinically used opioid agonists are relatively selective for mu receptors .

  Opioids exert their effects by selectively inhibiting the release of neurotransmitters such as acetylcholine, norepinephrine, dopamine, serotonin, and substance P.

  Pharmacologically, opioid agonists constitute an important class of drugs used for pain management. Unfortunately, the use of opioid agonists has the potential for abuse. In addition, oral administration of opioid agonists often results in significant first pass metabolism. Moreover, administration of opioid agonists results in significant central nervous system mediated effects such as respiratory depression, which can lead to death. Thus, the reduction of any one of these or another property will enhance their desirability as a therapeutic agent.

  The present invention seeks to address these and other needs in the prior art by providing (among other things) conjugates of water-soluble non-peptide oligomers and opioid agonists.

  In one or more embodiments of the present invention, a compound is provided that comprises an opioid agonist residue covalently bound to a water-soluble non-peptide oligomer (preferably via a stable bond).

  In one or more embodiments of the present invention, a compound is provided that comprises a residue of a kappa opioid agonist covalently linked to a water-soluble non-peptide oligomer (preferably via a stable linkage) [ Wherein the kappa opioid agonist is (i) preferentially selective for the kappa opioid receptor over both the mu opioid receptor and the delta opioid receptor within the same mammalian species; It should be understood that it has agonist activity at the receptor].

  In one or more embodiments of the present invention, a compound is provided that comprises a residue of a mu opioid agonist covalently linked (preferably via a stable linkage) to a water-soluble non-peptide oligomer [ Kappa opioid agonists can be preferentially selected for mu opioid receptors over (i) both kappa opioid receptors and delta opioid receptors within the same mammalian species, and (ii) agonist activity at mu receptors It should be understood that

式中、
Ｒ^１は、Ｈまたは［メチル、エチル、および−Ｃ（Ｏ）ＣＨ_３等の］有機ラジカルであり、
Ｒ^２は、ＨまたはＯＨであり、
Ｒ^３は、Ｈまたは有機ラジカルであり、
Ｒ^４は、Ｈまたは有機ラジカルであり、
点線（「−−−」）は、任意の二重結合を示し、
Ｙ^１は、Ｏ（酸素）またはＳであり、かつ
Ｒ^５は、（立体化学に関係なく）

から成る群より選択され、式中、Ｒ^６は、有機ラジカル［−Ｃ（Ｏ）ＣＨ_３を含む］である。 In one or more embodiments of the invention, a compound is provided, the compound comprising a residue of an opioid agonist covalently linked to a water-soluble non-peptide oligomer via a stable linkage, wherein the opioid agonist comprises: Having a structure encompassed by the following chemical formula:

Where
R ¹ is H or an organic radical [such as methyl, ethyl, and —C (O) CH ₃ ];
R ² is H or OH;
R ³ is H or an organic radical;
R ⁴ is H or an organic radical;
The dotted line ("---") indicates any double bond,
Y ¹ is O (oxygen) or S, and R ⁵ is (regardless of stereochemistry)

Wherein R ⁶ is an organic radical [including —C (O) CH ₃ ].

  In one or more embodiments of the present invention, a compound is provided, comprising a residue of an opioid agonist covalently linked to a water-soluble non-peptide oligomer via a stable or degradable linkage, wherein Opioid agonists include asimadoline, bremazosin, enadrine, ethylketocyclazocine, GR89,696, ICI204448, ICI197067, PD117,302, nalbuphine, pentazocine, quadazosin (WIN 44,441-3), salvinorin A, spiradrine, TRK-820, Selected from the group consisting of U50488 and U69593.

In one or more embodiments of the invention, a composition is provided, the composition comprising:
(I) a compound comprising a residue of an opioid agonist covalently bonded to a water-soluble non-peptide oligomer via a stable bond;
(Ii) optionally including pharmaceutically acceptable excipients.

  In one or more embodiments of the invention, a dosage form is provided, the dosage form comprising a compound comprising a residue of an opioid agonist covalently linked to a water-soluble non-peptide oligomer via a stable linkage.

  In one or more embodiments of the present invention, a method is provided that includes covalently coupling a water-soluble non-peptide oligomer to an opioid agonist.

  In one or more embodiments of the present invention, a method is provided comprising administering a compound comprising a residue of an opioid agonist covalently linked to a water-soluble non-peptide oligomer via a stable linkage. .

  In one or more embodiments of the invention, a method is provided, the method comprising binding (eg, selectively binding) a mu opioid receptor, wherein the binding comprises a water-soluble non-peptide. This is accomplished by administering a compound comprising an opioid agonist residue covalently linked to an oligomer. In one or more embodiments of the present invention, a method is provided, the method comprising binding (eg, selectively binding) a mu opioid receptor, wherein the binding comprises an effective amount of water solubility. This is accomplished by administering to a mammalian patient a compound comprising an opioid agonist residue covalently linked to a non-peptide oligomer.

  In one or more embodiments of the invention, a method is provided, the method comprising binding (eg, selectively binding) a kappa opioid receptor, wherein the binding comprises a water-soluble non-peptide. This is accomplished by administering a compound comprising an opioid agonist residue covalently linked to an oligomer. In one or more embodiments of the invention, a method is provided, the method comprising binding (eg, selectively binding) a kappa opioid receptor, wherein the binding comprises a water-soluble non-peptide oligomer. This is accomplished by administering to a mammalian patient a compound comprising an effective amount of an opioid agonist residue covalently linked to

  These and other objects, aspects, embodiments, and features of the invention will become more apparent when read in conjunction with the following detailed description.

FIG. 1 shows mu, kappa, and delta receptors over the parent molecular nalbuphine plotted as a function of PEG length for PEG-nalbuphine conjugates, as described in more detail in Example 4. It is a graph which shows the fold change of the binding affinity with respect to. As shown in FIG. 1, binding affinity decreases as a function of PEG chain length at mu opioid receptors and kappa opioid receptors, but not at delta opioid receptors, so that PEG conjugation 2 shows differentially affecting binding at the opioid receptor subtypes. Figures 2a and 2b are graphs showing the in vitro permeability and efflux ratios of various nalbuphine conjugates, as described in more detail in Example 9. These graphs show that (i) the permeability of PEG-nalbuphine conjugates in Caco-2 cells is reduced as a function of PEG chain length (Figure 2a), and (ii) PEG-nalbuphine conjugates are Shows that it is likely to be a substrate for (FIG. 2b). Figures 2a and 2b are graphs showing the in vitro permeability and efflux ratios of various nalbuphine conjugates, as described in more detail in Example 9. These graphs show that (i) the permeability of PEG-nalbuphine conjugates in Caco-2 cells is reduced as a function of PEG chain length (Figure 2a), and (ii) PEG-nalbuphine conjugates are Shows that it is likely to be a substrate for (FIG. 2b). FIG. 3 is a graph showing the brain: plasma ratio of various PEG-nalbuphine conjugates, as described in more detail in Example 10. This graph shows that PEG conjugates result in a decrease in the brain: plasma ratio of nalbuphine. FIG. 4 was administered in an analgesic assay to assess the percent of agony per n total number of mice in the study group and the degree of reduction or prevention of visceral pain in the mice, as described in detail in Example 18. it is a graph showing a dose of mPEG _n -O- morphine. Morphine was used as a control and the unconjugated parent molecule morphine sulfate was also administered to provide an additional reference point. The following conjugate series were evaluated: conjugates belonging to mPEG _2-7,9- O-morphine. FIG. 5 is administered in an analgesic assay to assess the percent of agony per n total number of mice in the study group and the degree of reduction or prevention of visceral pain in the mice, as described in detail in Example 18. is a graph showing a dose of mPEG _n -O- hydroxy codon conjugate. Morphine was used as a control and oxycodone, an unconjugated parent molecule, was also administered to provide an additional reference point. The following conjugate series were evaluated: conjugates belonging to PEG _1-4,6,7,9- O-hydroxycodone. FIG. 6 is administered in an analgesic assay to assess the percent of agony per n total number of mice in the study group and the degree of reduction or prevention of visceral pain in the mice, as described in detail in Example 18. it is a graph showing a dose of mPEG _n -O- codeine conjugates. Morphine was used as a control and the unconjugated parent molecule codeine was also administered to provide an additional reference point. The following conjugate series, namely conjugates belonging to mPEG _3-7,9- O-codeine were evaluated. 7-9 are plots showing the results of a hot plate latency analgesia assay in mice, as described in detail in Example 19. Specifically, the figure corresponds to a graph showing latency (time to lick hind legs) (seconds) and compound dose. FIG. 7 provides results for mPEG _1-5- O-hydroxycodone conjugate and unconjugated parent molecule, and FIG. 8 shows for mPEG _1-5- O-morphine conjugate and unconjugated parent molecule. Results are provided and FIG. 9 provides results for mPEG _2-5, 9-O-codeine conjugate and parent molecule. The presence of an asterisk beside the data point indicates p <0.05 for saline solutions by ANOVA / Dunnett. 7-9 are plots showing the results of a hot plate latency analgesia assay in mice, as described in detail in Example 19. Specifically, the figure corresponds to a graph showing latency (time to lick hind legs) (seconds) and compound dose. FIG. 7 provides results for mPEG _1-5- O-hydroxycodone conjugate and unconjugated parent molecule, and FIG. 8 shows for mPEG _1-5- O-morphine conjugate and unconjugated parent molecule. Results are provided and FIG. 9 provides results for mPEG _2-5, 9-O-codeine conjugate and parent molecule. The presence of an asterisk beside the data point indicates p <0.05 for saline solutions by ANOVA / Dunnett. 7-9 are plots showing the results of a hot plate latency analgesia assay in mice, as described in detail in Example 19. Specifically, the figure corresponds to a graph showing latency (time to lick hind legs) (seconds) and compound dose. FIG. 7 provides results for mPEG _1-5- O-hydroxycodone conjugate and unconjugated parent molecule, and FIG. 8 shows for mPEG _1-5- O-morphine conjugate and unconjugated parent molecule. Results are provided and FIG. 9 provides results for mPEG _2-5, 9-O-codeine conjugate and parent molecule. The presence of an asterisk beside the data point indicates p <0.05 for saline solutions by ANOVA / Dunnett. FIG. 10 shows compounds after intravenous administration of 1.0 mg / kg to rats, ie oxycodone (mPEG ₀ -oxycodone), mPEG ₁ -O-hydroxycodone, mPEG ₂ -O, as described in Example 21. - hydroxy codon, mPEG ₃ -O- hydroxy codon, mPEG ₄ -O- hydroxy codon, mPEG ₅ -O- hydroxy codon, mPEG ₆ -O- hydroxy codons, mPEG ₇ -O- hydroxy codon, and mPEG ₉ -O- Shown are mean (± standard deviation) plasma concentration-time profiles for hydroxycodone. FIG. 11 shows compounds after 5.0 mg / kg oral administration to rats, ie oxycodone (mPEG ₀ -oxycodone), mPEG ₁ -O-hydroxycodone, mPEG ₂ -O-, as described in Example 21. Hydroxy codon, mPEG ₃ -O-hydroxy codon, mPEG ₄ -O-hydroxy codon, mPEG ₅ -O-hydroxy codon, mPEG ₆ -O-hydroxy codon, mPEG ₇ -O-hydroxy codon, and mPEG ₉ -O-hydroxy Shown are mean (± standard deviation) plasma concentration-time profiles for codons. FIG. 12 shows compounds after intravenous administration of 1.0 mg / kg to rats, ie morphine (mPEG _{0 -morphine} ) and mPEG _1-7,9 -O-morphine, as described in detail in Example 22. Shown are mean (± standard deviation) plasma concentration-time profiles for conjugates. FIG. 13 shows compounds after 5.0 mg / kg intravenous administration to rats, ie morphine (mPEG _{0 -morphine} ) and mPEG _1-7,9- O-morphine conjugate, as described in Example 22. The mean (± standard deviation) plasma concentration-time characteristics for are shown. FIG. 14 shows compounds after intravenous administration of 1.0 mg / kg to rats, ie codeine (mPEG _{0 -codeine} ) and mPEG _1-7,9 -O-codeine, as described in detail in Example 23. Shown are mean (± standard deviation) plasma concentration-time profiles for conjugates. FIG. 15 shows compounds after 5.0 mg / kg intravenous administration to rats, ie codeine (mPEG _{0 -codeine} ) and mPEG _1-7,9 -O-codeine, as described in detail in Example 23. Shown are mean (± standard deviation) plasma concentration-time profiles for conjugates. FIGS. 16A, 16B, and 16C show various oligomeric mPEG _n —O-morphine, mPEG _n —O-codeine, and mPEG _n − after intravenous administration to rats, as described in Example 26. The brain: plasma ratio of the O-hydroxycodone conjugate is shown. The brain: plasma ratio of atenolol is provided in each figure as a basis for comparison. FIGS. 16A, 16B, and 16C show various oligomeric mPEG _n —O-morphine, mPEG _n —O-codeine, and mPEG _n − after intravenous administration to rats, as described in Example 26. The brain: plasma ratio of the O-hydroxycodone conjugate is shown. The brain: plasma ratio of atenolol is provided in each figure as a basis for comparison. FIGS. 16A, 16B, and 16C show various oligomeric mPEG _n —O-morphine, mPEG _n —O-codeine, and mPEG _n − after intravenous administration to rats, as described in Example 26. The brain: plasma ratio of the O-hydroxycodone conjugate is shown. The brain: plasma ratio of atenolol is provided in each figure as a basis for comparison. FIGS. 17A-17H show brain and plasma concentrations over time of morphine and various mPEG _n -O-morphine conjugates following intravenous administration to rats, as described in Example 27. Figure 17A (morphine, n = 0); Figure 17B (n = 1); Figure 17C (n = 2); Figure 17D (n = 3); Figure 17E (n = 4); Figure 17F (n = 5); FIG. 17G (n = 6); FIG. 17H (n = 7). FIGS. 17A-17H show brain and plasma concentrations over time of morphine and various mPEG _n -O-morphine conjugates following intravenous administration to rats, as described in Example 27. Figure 17A (morphine, n = 0); Figure 17B (n = 1); Figure 17C (n = 2); Figure 17D (n = 3); Figure 17E (n = 4); Figure 17F (n = 5); FIG. 17G (n = 6); FIG. 17H (n = 7) FIGS. 17A-17H show brain and plasma concentrations over time of morphine and various mPEG _n -O-morphine conjugates following intravenous administration to rats, as described in Example 27. Figure 17A (morphine, n = 0); Figure 17B (n = 1); Figure 17C (n = 2); Figure 17D (n = 3); Figure 17E (n = 4); Figure 17F (n = 5); FIG. 17G (n = 6); FIG. 17H (n = 7) FIGS. 17A-17H show brain and plasma concentrations over time of morphine and various mPEG _n -O-morphine conjugates following intravenous administration to rats, as described in Example 27. Figure 17A (morphine, n = 0); Figure 17B (n = 1); Figure 17C (n = 2); Figure 17D (n = 3); Figure 17E (n = 4); Figure 17F (n = 5); FIG. 17G (n = 6); FIG. 17H (n = 7) FIGS. 17A-17H show brain and plasma concentrations over time of morphine and various mPEG _n -O-morphine conjugates following intravenous administration to rats, as described in Example 27. Figure 17A (morphine, n = 0); Figure 17B (n = 1); Figure 17C (n = 2); Figure 17D (n = 3); Figure 17E (n = 4); Figure 17F (n = 5); FIG. 17G (n = 6); FIG. 17H (n = 7) FIGS. 17A-17H show brain and plasma concentrations over time of morphine and various mPEG _n -O-morphine conjugates following intravenous administration to rats, as described in Example 27. Figure 17A (morphine, n = 0); Figure 17B (n = 1); Figure 17C (n = 2); Figure 17D (n = 3); Figure 17E (n = 4); Figure 17F (n = 5); FIG. 17G (n = 6); FIG. 17H (n = 7) FIGS. 17A-17H show brain and plasma concentrations over time of morphine and various mPEG _n -O-morphine conjugates following intravenous administration to rats, as described in Example 27. Figure 17A (morphine, n = 0); Figure 17B (n = 1); Figure 17C (n = 2); Figure 17D (n = 3); Figure 17E (n = 4); Figure 17F (n = 5); FIG. 17G (n = 6); FIG. 17H (n = 7) FIGS. 17A-17H show brain and plasma concentrations over time of morphine and various mPEG _n -O-morphine conjugates following intravenous administration to rats, as described in Example 27. Figure 17A (morphine, n = 0); Figure 17B (n = 1); Figure 17C (n = 2); Figure 17D (n = 3); Figure 17E (n = 4); Figure 17F (n = 5); FIG. 17G (n = 6); FIG. 17H (n = 7) FIGS. 18A-18H show the brain and plasma concentrations over time of codeine and various mPEG _n —O-codeine conjugates following intravenous administration to rats, as described in Example 27. Figure 18A (codeine, n = 0); Figure 18B (n = 1); Figure 18C (n = 2); Figure 18D (n = 3); Figure 18E (n = 4); Figure 18F (n = 5); FIG. 18G (n = 6); FIG. 18H (n = 7). FIGS. 18A-18H show the brain and plasma concentrations over time of codeine and various mPEG _n —O-codeine conjugates following intravenous administration to rats, as described in Example 27. Figure 18A (codeine, n = 0); Figure 18B (n = 1); Figure 18C (n = 2); Figure 18D (n = 3); Figure 18E (n = 4); Figure 18F (n = 5); FIG. 18G (n = 6); FIG. 18H (n = 7). FIGS. 18A-18H show the brain and plasma concentrations over time of codeine and various mPEG _n —O-codeine conjugates following intravenous administration to rats, as described in Example 27. Figure 18A (codeine, n = 0); Figure 18B (n = 1); Figure 18C (n = 2); Figure 18D (n = 3); Figure 18E (n = 4); Figure 18F (n = 5); FIG. 18G (n = 6); FIG. 18H (n = 7). FIGS. 18A-18H show the brain and plasma concentrations over time of codeine and various mPEG _n —O-codeine conjugates following intravenous administration to rats, as described in Example 27. Figure 18A (codeine, n = 0); Figure 18B (n = 1); Figure 18C (n = 2); Figure 18D (n = 3); Figure 18E (n = 4); Figure 18F (n = 5); FIG. 18G (n = 6); FIG. 18H (n = 7). FIGS. 18A-18H show the brain and plasma concentrations over time of codeine and various mPEG _n —O-codeine conjugates following intravenous administration to rats, as described in Example 27. Figure 18A (codeine, n = 0); Figure 18B (n = 1); Figure 18C (n = 2); Figure 18D (n = 3); Figure 18E (n = 4); Figure 18F (n = 5); FIG. 18G (n = 6); FIG. 18H (n = 7). FIGS. 18A-18H show the brain and plasma concentrations over time of codeine and various mPEG _n —O-codeine conjugates following intravenous administration to rats, as described in Example 27. Figure 18A (codeine, n = 0); Figure 18B (n = 1); Figure 18C (n = 2); Figure 18D (n = 3); Figure 18E (n = 4); Figure 18F (n = 5); FIG. 18G (n = 6); FIG. 18H (n = 7). FIGS. 18A-18H show the brain and plasma concentrations over time of codeine and various mPEG _n —O-codeine conjugates following intravenous administration to rats, as described in Example 27. Figure 18A (codeine, n = 0); Figure 18B (n = 1); Figure 18C (n = 2); Figure 18D (n = 3); Figure 18E (n = 4); Figure 18F (n = 5); FIG. 18G (n = 6); FIG. 18H (n = 7). FIGS. 18A-18H show the brain and plasma concentrations over time of codeine and various mPEG _n —O-codeine conjugates following intravenous administration to rats, as described in Example 27. Figure 18A (codeine, n = 0); Figure 18B (n = 1); Figure 18C (n = 2); Figure 18D (n = 3); Figure 18E (n = 4); Figure 18F (n = 5); FIG. 18G (n = 6); FIG. 18H (n = 7). FIGS. 19A-19H show brain and plasma concentrations over time of oxycodone and various mPEGn-O-hydroxycodone conjugates following intravenous administration to rats, as described in Example 27. Figure 19A (oxycodone, n = 0); Figure 19B (n = 1); Figure 19C (n = 2); Figure 19D (n = 3); Figure 19E (n = 4); Figure 19F (n = 5); FIG. 19G (n = 6); FIG. 19H (n = 7). FIGS. 19A-19H show brain and plasma concentrations over time of oxycodone and various mPEGn-O-hydroxycodone conjugates following intravenous administration to rats, as described in Example 27. Figure 19A (oxycodone, n = 0); Figure 19B (n = 1); Figure 19C (n = 2); Figure 19D (n = 3); Figure 19E (n = 4); Figure 19F (n = 5); FIG. 19G (n = 6); FIG. 19H (n = 7). FIGS. 19A-19H show brain and plasma concentrations over time of oxycodone and various mPEGn-O-hydroxycodone conjugates following intravenous administration to rats, as described in Example 27. Figure 19A (oxycodone, n = 0); Figure 19B (n = 1); Figure 19C (n = 2); Figure 19D (n = 3); Figure 19E (n = 4); Figure 19F (n = 5); FIG. 19G (n = 6); FIG. 19H (n = 7). FIGS. 19A-19H show brain and plasma concentrations over time of oxycodone and various mPEGn-O-hydroxycodone conjugates following intravenous administration to rats, as described in Example 27. Figure 19A (oxycodone, n = 0); Figure 19B (n = 1); Figure 19C (n = 2); Figure 19D (n = 3); Figure 19E (n = 4); Figure 19F (n = 5); FIG. 19G (n = 6); FIG. 19H (n = 7). FIGS. 19A-19H show brain and plasma concentrations over time of oxycodone and various mPEGn-O-hydroxycodone conjugates following intravenous administration to rats, as described in Example 27. Figure 19A (oxycodone, n = 0); Figure 19B (n = 1); Figure 19C (n = 2); Figure 19D (n = 3); Figure 19E (n = 4); Figure 19F (n = 5); FIG. 19G (n = 6); FIG. 19H (n = 7). FIGS. 19A-19H show brain and plasma concentrations over time of oxycodone and various mPEGn-O-hydroxycodone conjugates following intravenous administration to rats, as described in Example 27. Figure 19A (oxycodone, n = 0); Figure 19B (n = 1); Figure 19C (n = 2); Figure 19D (n = 3); Figure 19E (n = 4); Figure 19F (n = 5); FIG. 19G (n = 6); FIG. 19H (n = 7). FIGS. 19A-19H show brain and plasma concentrations over time of oxycodone and various mPEGn-O-hydroxycodone conjugates following intravenous administration to rats, as described in Example 27. Figure 19A (oxycodone, n = 0); Figure 19B (n = 1); Figure 19C (n = 2); Figure 19D (n = 3); Figure 19E (n = 4); Figure 19F (n = 5); FIG. 19G (n = 6); FIG. 19H (n = 7). FIGS. 19A-19H show brain and plasma concentrations over time of oxycodone and various mPEGn-O-hydroxycodone conjugates following intravenous administration to rats, as described in Example 27. Figure 19A (oxycodone, n = 0); Figure 19B (n = 1); Figure 19C (n = 2); Figure 19D (n = 3); Figure 19E (n = 4); Figure 19F (n = 5); FIG. 19G (n = 6); FIG. 19H (n = 7).

  As used herein, the singular forms “a”, “an”, and “the” include plural referents unless the context clearly dictates otherwise.

  In describing and claiming the present invention, the following terminology will be used in accordance with the definitions set forth below.

  “Water-soluble non-peptide oligomer” refers to an oligomer having solubility in water at room temperature that is at least 35% by weight, preferably greater than 70% by weight, more preferably greater than 95% by weight. In general, an unfiltered aqueous preparation of “water-soluble” oligomers transmits at least 75%, more preferably at least 95%, of the amount of light transmitted by the same solution after filtering. However, it is most preferred that the water-soluble oligomer is soluble in at least 95% by weight water or completely soluble in water. With respect to “non-peptide”, an oligomer is non-peptide when it has less than 35% by weight amino acid residues.

  The terms “monomer”, “monomer subunit”, and “monomer unit” are used interchangeably herein and refer to one of the basic structural units of a polymer or oligomer. In the case of a homo-oligomer, a single repeating structural unit forms the oligomer. In the case of a co-oligomer, two or more structural units are repeated (in a pattern or randomly) to form the oligomer. Preferred oligomers used in connection with the present invention are homo-oligomers. Water-soluble non-peptide oligomers generally comprise one or more monomers joined together so as to form a monomer chain. Oligomers can be formed from a single monomer type (ie, a homo-oligomer) or from two or three monomer types (ie, are co-oligomers).

  An “oligomer” is a molecule having from about 2 to about 50 monomers, preferably from about 2 to about 30 monomers. The composition of the oligomer can vary. Specific oligomers used in the present invention include those having various shapes such as straight lines, branches, and forks as described in detail below.

“PEG” or “polyethylene glycol” as used herein is intended to encompass any water-soluble poly (ethylene oxide). Unless otherwise specified, a “PEG oligomer” (also referred to as oligoethylene glycol) is one in which substantially all (and more preferably all) monomer subunits are ethylene oxide subunits. However, the oligomer may contain distinct end capping or functional groups, for example for conjugation. In general, PEG oligomers for use in the present invention are represented by “— (CH ₂ CH ₂ O) _n —” or “— (CH ₂ CH ₂ O, for example, based on whether the terminal oxygen was replaced during the synthetic transformation. _N-1 CH ₂ CH ₂ — ”. For PEG oligomers, “n” varies from about 2 to 50, preferably from about 2 to 30, and the configuration of the end groups and the overall PEG can vary. When PEG further comprises, for example, a functional group A for linking to a small molecule drug, when the functional group is attached to the PEG oligomer, (i) an oxygen-oxygen bond (—O—O—, peroxide bond) ), Or (ii) does not result in the formation of nitrogen-oxygen bonds (NO, ON).

An “end capping group” is generally a group containing a non-reactive carbon attached to the terminal oxygen of the PEG oligomer. Exemplary end capping groups include C _1-5 alkyl groups such as methyl, ethyl, and benzyl, and aryl, heteroaryl, cyclo, heterocyclo, and the like. For the purposes of the present invention, suitable capping groups have a relatively low molecular weight such as methyl or ethyl. The end capping group can also include a detectable label. Such labels include, but are not limited to, luminescent materials, chemiluminescent materials, moieties used for enzyme labeling, colorimetric labels (eg, dyes), metal ions, and radioactive moieties.

  “Branched” refers to an oligomer having two or more polymers that represent distinct arms “arms” that extend from the branch point with respect to the shape or overall structure of the oligomer.

  “Forked” refers to an oligomer having two or more functional groups extending from a branch point (generally through one or more atoms) with respect to the shape or overall structure of the oligomer.

  A “branch point” refers to a branch point that includes one or more atoms where an oligomer branches or forks from a linear structure into one or more additional arms.

  The term “reactive” or “active” refers to a functional group that reacts easily or at a practical rate under conventional conditions of organic synthesis. This is in contrast to groups that do not react or require strong catalysts or impractical conditions to react (ie, “non-reactive” or “inert” groups).

  “Non-reactive” refers to the functional groups present on the molecules in the reaction mixture, largely in the intact state under conditions where the groups are effective to produce the desired reaction in the reaction mixture. Indicates that

  A “protecting group” is a moiety that prevents or prevents the reaction of a particular chemically reactive functional group in a molecule under certain reaction conditions. Protecting groups depend on the type of chemically reactive group being protected and the reaction conditions used and the presence of additional reactive or protecting groups in the molecule. Examples of functional groups that can be protected include carboxylic acid groups, amino groups, hydroxyl groups, thiol groups, carbonyl groups, and the like. Typical protecting groups for carboxylic acids include esters (such as p-methoxybenzyl ester), amides, and hydrazides, for amino groups, carbamates (such as tert-butoxycarbonyl) and amides, for hydroxyl groups. , Ethers and esters, thiol groups for thioethers and thioesters, carbonyl groups for acetals and ketals, and the like. Such protecting groups are well known to those skilled in the art and are described, for example, in T.W. W. Green and G.G. M.M. Wuts, Protecting Groups in Organic Synthesis, Third Edition, Wiley, New York, 1999 and references cited therein.

  A functional group in “protected form” refers to a functional group bearing a protecting group. As used herein, “functional group” or any synonym thereof encompasses protected forms thereof.

  A “physiologically cleavable”, “hydrolyzable” or “degradable” bond is a relatively unstable bond that reacts with water (ie, is hydrolyzed) under normal physiological conditions. is there. Under normal physiological conditions, the tendency to bind to hydrolyze in water is not only due to the general type of bond connecting two central atoms, but also to the substituents attached to these central atoms. Also depends. Such coupling is generally recognizable by those skilled in the art. Suitable hydrolytically unstable or weak bonds include carboxylic esters, phosphate esters, anhydrides, acetals, ketals, acyloxyalkyl ethers, imines, orthoesters, peptides, oligonucleotides, thioesters, and carbonates. For example, but not limited to.

  “Enzymolytic bond” means a bond that undergoes degradation by one or more enzymes under normal physiological conditions.

  A “stable” bond or bond is a chemical moiety or a moiety that is substantially stable in water, i.e., does not undergo hydrolysis under normal physiological conditions to any detectable extent over an extended period of time. Refers to a bond, typically a covalent bond. Examples of hydrolytically stable bonds include, but are not limited to, carbon-carbon bonds (eg, in aliphatic chains), ethers, amides, urethanes, amines, and the like. In general, a stable bond is one that exhibits a rate of hydrolysis of less than about 1-2% per day under normal physiological conditions. Typical chemical bond hydrolysis rates can be found in most standard chemical books.

  In the context of describing the consistency of oligomers in a given composition, “substantially” or “basically” means almost completely or completely, for example, within a given quantity 95% or more, more preferably 97% or more, still more preferably 98% or more, still more preferably 99% or more, still more preferably 99.9% or more, and most preferably 99.99% or more.

  “Monodisperse” refers to an oligomeric composition in which substantially all oligomers in the composition have a well-defined single molecular weight and a defined number of monomers as defined by chromatography or mass spectrometry. A monodisperse oligomer composition is pure in a sense. That is, it includes molecules having a substantially single, definable number of monomers, rather than several different numbers of monomers (ie, oligomer compositions having three or more different oligomer sizes). The monodisperse oligomer composition has a MW / Mn value of 1.0005 or less, more preferably a MW / Mn value of 1.0000. Thus, a composition consisting of a monodisperse conjugate has a single (as an integer) definable number of monomers in which substantially all oligomers of all conjugates in the composition are not distributed, When the oligomer is not conjugated to the opioid agonist residue, it is meant to have a MW / Mn value of 1.0005, more preferably a MW / Mn value of 1.0000. However, a composition comprising a monodisperse conjugate can include one or more non-conjugate substances such as solvents, reagents, excipients, and the like.

  “Bimodal” refers to an oligomer composition in which substantially all oligomers in the composition have a different number of monomers that can be defined (as an integer) in one of the two, rather than distributed. And refers to an oligomeric composition whose molecular weight distribution appears as two separate distinguishable peaks when plotted as fractions versus molecular weight. For the bimodal oligomer compositions described herein, the two peaks may be different, but it is generally preferred that each peak be symmetric with respect to its average. Ideally, the polydispersity index Mw / Mn of each peak in the bimodal distribution is 1.01 or less, more preferably 1.001 or less, still more preferably 1.0005 or less, and most preferably 1.0000. MW / Mn value. Thus, a composition consisting of a bimodal conjugate has a different number of substantially definable oligomers of all conjugates in the composition, not a large distribution, which can be defined as one (as an integer) of two. MW / Mn value of 1.01 or less, more preferably 1.001 or less, even more preferably 1.0005 or less, most preferably when the monomer is present and the oligomer is not bound to the opioid agonist residue. It means having a MW / Mn value of 1.0000. However, a composition comprising a bimodal conjugate can include one or more non-conjugate substances such as solvents, reagents, excipients, and the like.

  An “opioid agonist” as used herein is an organic compound, inorganic, having a molecular weight typically less than about 1000 daltons (and typically less than 500 daltons) and having some activity as a mu and / or kappa agonist. Used broadly to refer to a compound or organometallic compound. Opioid agonists include oligopeptides and other biomolecules having a molecular weight of less than about 1000.

  A “biological membrane” is typically any membrane made from specialized cells or tissues and functions as a barrier to at least some foreign or other undesirable substances. As used herein, a “biological membrane” includes a membrane associated with a physiological protective barrier, such as the blood brain barrier (BBB), blood cerebrospinal fluid barrier, blood placental barrier, blood milk barrier, blood Includes the testicular barrier and mucosal barriers including the vaginal mucosa, urethral mucosa, anal mucosa, buccal mucosa, sublingual mucosa, rectal mucosa and the like. Unless the context clearly states otherwise, the term “biological membrane” does not include membranes associated with the intermediate gastrointestinal tract (eg, stomach and small intestine).

  “Biomembrane crossing rate”, as used herein, provides a measure of the ability of a compound to cross a biological membrane (such as a membrane associated with the blood brain barrier). Various methods can be used to assess the transport of molecules across any given biological membrane. Methods for assessing the biological membrane crossing rate associated with any given biological barrier (eg, blood cerebrospinal fluid barrier, blood placental barrier, blood milk barrier, intestinal barrier, etc.) are known in the art. And / or are described herein and / or in the relevant literature and / or can be determined by one skilled in the art.

  “Reduced metabolic rate” means, for the present invention, small molecules of water-soluble oligomers compared to the rate of metabolism of small molecule drugs that are not bound to water-soluble oligomers (ie, small molecule drugs themselves) or reference standard materials. Refers to a measurable decrease in the metabolic rate of a molecular drug conjugate. In the special case of “decrease in the first pass rate of metabolism”, the “decrease in metabolic rate” is also the same except that the small molecule drug (or reference standard material) and the corresponding conjugate are administered orally. is necessary. Orally administered drugs are absorbed from the gastrointestinal tract into the portal circulation and must pass through the liver before reaching the systemic circulation. Since the liver is the main site of drug metabolism or biotransformation, a significant amount of drug can be metabolized before reaching the systemic circulation. The degree of first-pass metabolism, and therefore any decrease in it, can be measured in many different ways. For example, animal blood samples can be collected at defined intervals for metabolite levels, and plasma or serum is analyzed by liquid chromatography / mass spectrometry. Other techniques for measuring “reduction in metabolic rate” associated with first pass metabolism and other metabolic processes are known in the art and are described herein and / or in the relevant literature, and Or can be determined by one skilled in the art. The conjugates of the invention have at least one of the values of at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, and at least about 90%. Preferably, it can provide a decrease in metabolic rate that satisfies. “Orally bioavailable” compounds (such as small molecule drugs or conjugates thereof) preferably have a bioavailability of greater than 25%, preferably greater than 70%, when administered orally. And the bioavailability of the compound is the fraction of the administered drug that reaches the systemic circulation in a non-metabolic form.

  “Alkyl” refers to a hydrocarbon chain, typically ranging from about 1 to 20 atoms in length. Such hydrocarbon chains are preferably, but not necessarily, saturated and may be branched or straight chain, but generally straight chain is preferred. Exemplary alkyl groups include methyl, ethyl, propyl, butyl, pentyl, 1-methylbutyl, 1-ethylpropyl, 3-methylpentyl and the like. As used herein, “alkyl” includes cycloalkyl when three or more carbon atoms are referred to. An “alkenyl” group is an alkyl of 2 to 20 carbon atoms with at least one carbon-carbon double bond.

“Substituted alkyl” or “substituted C _qr alkyl” in which q and r are integers representing the range of carbon atoms contained in the alkyl group is one, two, or three halo (eg, F, Cl, Br, I), trifluoromethyl, hydroxy, C _1-7 alkyl (eg, methyl, ethyl, n-propyl, isopropyl, butyl, t-butyl, etc.), C _1-7 alkoxy, C _1-7 acyloxy, C _3-7 means an alkyl group as described above substituted by heterocycle, amino, phenoxy, nitro, carboxy, carboxy, acyl, cyano. A substituted alkyl group can be substituted once, twice, or three times with the same or different substituents.

  “Lower alkyl” refers to an alkyl group containing from 1 to 6 carbon atoms, and may be linear or branched, as exemplified by methyl, ethyl, n-butyl, i-butyl, t-butyl. Good. “Lower alkenyl” refers to a lower alkyl group of 2 to 6 carbon atoms having at least one carbon-carbon double bond.

  A “non-interfering substituent” is a group that, when present in a molecule, is generally nonreactive with another functional group contained within the molecule.

“Alkoxy” refers to the group —O—R, where R is alkyl or substituted alkyl, preferably C ₁ -C ₂₀ alkyl (eg, methoxy, ethoxy, propyloxy, benzyl, etc.), preferably C it is a ₁ -C _7.

  “Pharmaceutically acceptable excipient” or “pharmaceutically acceptable carrier” refers to an ingredient that can be included in a composition of the invention, as compared to a composition lacking that ingredient, It is an object to provide a composition that has advantages (eg, more suitable for administration to a patient) and that is recognized as not producing a significant toxicological effect on the patient.

The term “aryl” means an aromatic group having up to 14 carbon atoms. Aryl groups include phenyl, naphthyl, biphenyl, phenanthrenyl, naphthacenyl and the like. “Substituted phenyl” and “substituted aryl” are each halo (F, Cl, Br, I) hydroxy, hydroxy, cyano, nitro, alkyl (eg, C _1-6 alkyl), alkoxy (eg, C _1-6 alkoxy) ), Benzyloxy, carboxy, aryl, etc., substituted by 1, 2, 3, 4, or 5 (eg 1-2, 1-3, or 1-4 substituents) Meaning a phenyl group and an aryl group.

  An “aromatic-containing moiety” is a group of atoms including at least aryl and optionally one or more atoms. Suitable aromatic-containing moieties are described herein.

For the sake of brevity, chemical moieties are defined throughout and referred to primarily as monovalent chemical moieties (eg, alkyl, aryl, etc.). However, such terms are also used to convey the corresponding multivalent moiety in an appropriate structural environment that will be apparent to those skilled in the art. For example, an “alkyl” moiety generally refers to a monovalent radical (eg, CH ₃ —CH ₂ —), although in certain circumstances a divalent link moiety can be an “alkyl” In those instances, one of ordinary skill in the art will understand that alkyl becomes a divalent radical (eg, —CH ₂ —CH ₂ —) and is equivalent to the term “alkylene”. (Similarly, in the context where a divalent moiety is required and described as “aryl”, one skilled in the art will understand that the term “aryl” refers to arylene, the corresponding divalent moiety. ). All atoms have the usual number of valence electrons for bond formation (ie, 4 for carbon, 3 for nitrogen, 2 for oxygen, and 2 for sulfur based on the oxidation state of sulfur. 4 or 6).

  A “pharmacologically effective amount”, “physiologically effective amount”, and “therapeutically effective amount” are necessary to provide a threshold level of an active agent and / or conjugate in the bloodstream or in a target tissue. , Used interchangeably herein to mean the amount of water-soluble oligomeric small molecule drug conjugate present in the composition. The exact amount will depend on many factors such as, for example, the particular active agent, the composition and physical properties of the composition, the subject patient population, patient considerations, etc., and the information provided herein and related documents It can be easily determined by one skilled in the art based on the information available therein.

  A “bifunctional” oligomer is an oligomer that contains two functional groups therein, typically at its ends. When the functional groups are the same, the oligomer is said to be homobifunctional. When the functional groups are different, the oligomer is said to be heterobifunctional.

  Basic or acidic reactants described herein include those that are neutral and charged, and any corresponding salt forms thereof.

  The term “patient” generally refers to a condition that, although not necessarily, can be prevented or treated by administration of a conjugate described herein in the form of a water-soluble oligomeric small molecule drug conjugate. Refers to living organisms suffering from or prone to the condition, including both humans and animals.

  “Optional” or “optionally” means that the situation described subsequently may occur, but does not necessarily occur, and the description includes when the situation occurs and when it does not occur means.

  As mentioned above, the present invention is directed to compounds comprising opioid agonist residues covalently linked to water-soluble non-peptide oligomers via (among other things) stable or degradable linkages.

に包含される構造を有し、
式中、
Ｒ^１は、Ｈまたは［メチル、エチル、および−Ｃ（Ｏ）ＣＨ_３等の］有機ラジカルであり、
Ｒ^２は、ＨまたはＯＨであり、
Ｒ^３は、Ｈまたは有機ラジカルであり、
Ｒ^４は、Ｈまたは有機ラジカルであり、
点線（「−−−」）は、任意の二重結合を示し、
Ｙ^１は、ＯまたはＳであり、かつ
Ｒ^５は、（立体化学に関係なく）

から成る群より選択され、Ｒ^６は、有機ラジカル［Ｃ（Ｏ）ＣＨ_３を含む］である。例示的なＲ^３基には、メチル、エチル、イソプロピル、およびそれらに類するものなどの低級アルキル、ならびに以下：

を含む。 In one or more embodiments of the present invention, a compound is provided that comprises a residue of an opioid agonist covalently linked to a water-soluble non-peptide oligomer via a stable or degradable linkage, wherein the opioid An agonist has the following chemical formula:

Having a structure encompassed by
Where
R ¹ is H or an organic radical [such as methyl, ethyl, and —C (O) CH ₃ ];
R ² is H or OH;
R ³ is H or an organic radical;
R ⁴ is H or an organic radical;
The dotted line ("---") indicates any double bond,
Y ¹ is O or S and R ⁵ is (regardless of stereochemistry)

R ⁶ is an organic radical [including C (O) CH ₃ ]. Exemplary R ³ groups include lower alkyls such as methyl, ethyl, isopropyl, and the like, and the following:

including.

によって包含される構造を有し、式中、
Ｎ^＊は、窒素であり、
Ａｒは、シクロヘキシル、フェニル、ハロフェニル、メトキシフェニル、アミノフェニル、ピリジル、フリル、およびチエニルから成る群より選択され、
Ａｌｋは、エチレンおよびプロピレンから成る群より選択され、
Ｒ_ＩＩは、低級アルキル、低級アルコキシ、ジメチルアミノ、シクロプロピル、１−ピロリジル、モルフォリノ（好ましくは、エチルなどの低級アルキル）から成る群より選択され、
Ｒ_ＩＩ’は、水素、メチル、およびメトキシからなる群より選択され、かつ
Ｒ_ＩＩ”は、水素および有機ラジカル（好ましくは低級アルキル）からなる群より選択される。
化学式ＩＩに関して、条件に応じて、アミンのうちの１つまたは両方（しかしより典型的には、化学式ＩＩにおいて星印で標識されたアミン（「Ｎ^＊」））をプロトン化することができる。 In one or more embodiments of the present invention, a compound is provided, comprising a residue of an opioid agonist covalently linked to a water soluble non-peptide oligomer via a stable or degradable linkage, wherein The opioid agonist has the following formula:

Having the structure encompassed by:
N ^* is nitrogen,
Ar is selected from the group consisting of cyclohexyl, phenyl, halophenyl, methoxyphenyl, aminophenyl, pyridyl, furyl, and thienyl;
Alk is selected from the group consisting of ethylene and propylene;
R _II is selected from the group consisting of lower alkyl, lower alkoxy, dimethylamino, cyclopropyl, 1-pyrrolidyl, morpholino (preferably lower alkyl such as ethyl);
R _II ′ is selected from the group consisting of hydrogen, methyl, and methoxy, and R _II ″ is selected from the group consisting of hydrogen and an organic radical (preferably lower alkyl).
With respect to Formula II, depending on the conditions, one or both of the amines (but more typically the amine labeled with an asterisk (“N ^* ”) in Formula II) can be protonated.

  Specific examples of opioid agonists include acetorphine, acetyldihydrocodeine, acetyldihydrocodeinone, acetylmorphinone, alfentanil, allylprozin, alphaprozin, anilellidine, benzylmorphine, vegitramide, buprenorphine, butorphanol, clonitazene, codeine , Desomorphin, Dextromoramide, Dezocine, Diampromide, Diamorphin, Dihydrocodeine, Dihydromorphine, Dimenoxadol, Dimefeptanol, Dimethylthianbutene, Dioxafetilbutyrate, Dipipanone, Epeptazosin, Ethoheptadine, Ethylmethylthianbutene, Ethylmorphine , Etonitazen, etorphine, dihydroethorphine, fentanyl and derivatives, heroy , Hydrocodone, hydroxycodone, hydromorphone, hydroxypetidin, isomethadone, ketobemidone, levorphanol, levofenacil morphane, lofentanil, meperidine, meptazinol, metazosin, methadone, methopone, morphine, mirophine, narcein, nicomorphine, norlevorphanol , Normesadone, nalolphine, nalbuphine, normorphin, norpipanone, opium, oxycodone, oxymorphone, papaveretam, pentazocine, phenadoxone, phenomorphan, phenazosin, phenoperidine, pinomidine, pyritramide, proheptadine, promedol, propridine, propoxyphene, sufentanil, And tramadol. In certain embodiments, the opioid agonist is hydrocodone, morphine, hydromorphone, oxycodone, codeine, levorphanol, meperidine, methadone, oxymorphone, buprenorphine, fentanyl, dipipanone, heroin, tramadol, nalbuphine, etorphine, dihydroethorphine, butorphanol, Selected from the group consisting of levorphanol.

  The advantages of the compounds of the present invention are their ability to reduce metabolism and / or to retain some opioid agonist activity while resulting in reduced central nervous system mediated effects associated with the unconjugated form of the corresponding opioid agonist. It is thought that. While not wishing to be bound by theory, the oligomer-containing conjugates described herein reduce the overall affinity of the compound for substrates on which the oligomer can metabolize opioid agonists. Because it serves a role, it is not likely to be readily metabolized (in contrast to the unconjugated “original” opioid agonist). In addition (again, not wishing to be bound by theory), the additional magnitude introduced by the oligomers traverses the blood brain barrier (in contrast to the unconjugated “original” opioid agonist) Reduce the ability of the compound.

  Use of oligomers to form conjugates of the invention (eg, from monodisperse or bimodal compositions of oligomers, as opposed to relatively impure compositions) associated with the corresponding small molecule drugs Certain properties can be advantageously changed. For example, the conjugates of the present invention can penetrate the blood brain barrier when administered by any of a number of suitable routes of administration, such as parenteral, oral, transdermal, buccal, pulmonary, or nasal. It exhibits a decline in sex. Conjugates exhibit slow, minimal blood-brain barrier crossing or virtually do not pass through it, but nevertheless if systemic circulation is intended through the gastrointestinal (GI) wall when intended for oral delivery It is preferable to enter. Furthermore, the conjugates of the present invention maintain some bioactivity and bioavailability in their conjugated form as compared to the bioactivity and bioavailability of all oligomer-free compounds.

  With respect to the blood brain barrier (“BBB”), this barrier limits the transfer of drugs from the blood to the brain. This barrier consists of a continuous layer of unique endothelial cells connected by tight junctions. The brain capillaries, which account for more than 95% of the total surface area of the BBB, are the main route of entry into the central nervous system for most solutes and drugs.

  For compounds where the degree of blood-brain barrier crossing ability is not readily known, such ability may be determined using a suitable animal model, such as the in situ rat brain perfusion (“RBP”) model described herein. Can be determined. Briefly, the RBP method includes cannulation of the carotid artery, followed by perfusion with a compound solution under controlled conditions, followed by a flushing step to remove the compound remaining in the vascular gap. (Such analyzes can be performed by contract research institutions such as Absorption Systems, Exton, PA, for example). More specifically, in the RBP model, a cannula is placed in the left carotid artery and the side branch is tied. Physiological buffer containing the analyte (typically but not necessarily at a concentration level of 5 micromolar) is perfused at a flow rate of about 10 mL / min in a single pass perfusion experiment. After 30 seconds, the perfusion is stopped and the cerebrovascular contents are washed away with a compound-free buffer for another 30 seconds. Thereafter, the brain tissue is removed, and the compound concentration is analyzed by liquid chromatography using tandem mass spectrometry detection (LC / MS / MS). Alternatively, blood brain barrier permeability is a calculation of the molecular polar surface area (“PSA”) of a compound, defined as the sum of the surface contributions of polar atoms (usually oxygen, nitrogen, and bonded hydrogen) in the molecule. And can be estimated. PSA has been shown to correlate with compound transport properties such as blood brain barrier transport. Methods for determining the PSA of compounds are described, for example, in Ertl, P. et al. , Et al. , J .; Med. Chem. 2000, 43, 3714-3717, and Kelder, J. et al. , Et al. Pharm. Res. 1999, 16, 1514-1519.

  With respect to the blood brain barrier, water-soluble non-peptide oligomer-small molecule drug conjugates exhibit a reduced blood-brain barrier crossing rate compared to the cross-linking rate of small molecule drugs that are not bound to water-soluble non-peptide oligomers. Preferred exemplary reductions in blood brain barrier crossing rate of the compounds described herein include at least about 30%, as compared to the blood brain barrier crossing rate of a small molecule drug that is not bound to a water soluble oligomer. A reduction of about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, or at least about 90%. The preferred decrease in blood brain barrier crossing rate for the conjugate is at least about 20%.

  As mentioned above, the compounds of the present invention comprise residues of opioid agonists. Assay methods for determining whether a given compound can function as an agonist at the mu receptor or kappa receptor (regardless of whether the compound is in conjugated form) are described below.

  In some cases, opioid agonists can be obtained from commercial sources. In addition, opioid agonists can be obtained through chemical synthesis. Synthetic techniques for preparing opioid agonists are described in the literature and, for example, U.S. Pat. Nos. 2,628,962, 2,654,756, 2,649,454, and 2,806,033. It is described in.

  Each of these (and other) opioid agonists can be covalently bound to the water-soluble non-peptide oligomer (directly or through one or more atoms).

  Small molecule drugs useful in the present invention generally have a molecular weight of less than 1000 Da. Exemplary molecular weights for small molecule drugs include less than about 950, less than about 900, less than about 850, less than about 800, less than about 750, less than about 700, less than about 650, less than about 600, less than about 550, less than about 500. , Less than about 450, less than about 400, less than about 350, and less than about 300 molecular weight.

  In the chiral case, the small molecule drug used in the present invention can be a racemic mixture, or an optically active form such as a single optically active enantiomer, or any combination or ratio of enantiomers (ie, a scalemic mixture). In addition, small molecule drugs may have one or more geometric isomers. With respect to geometric isomers, the composition can include a single geometric isomer or a mixture of two or more geometric isomers. The small molecule drugs used in the present invention can be in their conventional active form or may have some modification. For example, a small molecule drug may have a target agent, tag, or transporter attached thereto, before or after the covalent attachment of the oligomer. Alternatively, the small molecule drug is conjugated to it, such as a phospholipid (eg, distearoylphosphatidylethanolamine, ie “DSPE”, or dipalmitoyl diphosphatidylethanolamine, ie “DPPE”), or a small fatty acid. Can have a lipophilic portion. In some cases, however, it is preferred that the small molecule drug moiety does not contain a linkage to the lipophilic moiety.

  Opioid agonists for attachment to water-soluble non-peptide oligomers have free hydroxyl, carboxyl, thio, amino groups, etc. (ie, “handles”) suitable for covalent attachment to the oligomer. In addition, an opioid agonist converts a reactive group, preferably from one of its existing functional groups, into a functional group suitable for forming a stable covalent bond between the oligomer and the drug. Can be modified.

  Thus, each oligomer comprises an alkylene oxide such as ethylene oxide or propylene oxide, an olefin alcohol such as vinyl alcohol, 1-propenol or 2-propenol, vinyl pyrrolidone, preferably a hydroxyalkylmethacrylamide or hydroxyalkyl methacrylate wherein alkyl is methyl, Consists of up to three different monomer types selected from the group consisting of α-hydroxy acids such as lactic acid or glycolic acid, phosphazenes, oxazolines, amino acids, monosaccharides, sugars, carbohydrates such as mannitol, and N-acryloylmorpholine Is done. Suitable monomer types include alkylene oxides, olefinic alcohols, hydroxyalkyl methacrylamides or methacrylates, N-acryloylmorpholine, and α-hydroxy acids. Each oligomer is independently preferably a co-oligomer composed of two types of monomers selected from this group, or more preferably a homo-oligomer composed of one type of monomer selected from this group. .

  The two types of monomers in the co-oligomer can be the same type of monomers, for example, two alkylene oxides such as ethylene oxide and propylene oxide. The oligomer is preferably an ethylene oxide homo-oligomer. Usually, but not necessarily, the end (or ends) of the oligomer that is not covalently linked to the small molecule is sealed to render it inactive. Alternatively, the termini may contain reactive groups. When the termini are reactive groups, the reactive groups are protected so that they are inactive, or as necessary, under the conditions of final oligomer formation or during covalent attachment of the oligomer to the small molecule drug. Selected. One commonly used terminal functional group is hydroxyl or —OH, particularly for oligoethylene oxide.

  Water soluble non-peptide oligomers (eg, “POLY” in various structures provided herein) can have any of a number of different shapes. For example, it can be straight, branched or forked. Most typically, water-soluble non-peptide oligomers are straight or branched, for example with one branch point. Although much of the discussion herein focuses on poly (ethylene oxide) as an exemplary oligomer, the discussion and structure presented herein includes any of the water-soluble non-peptide oligomers described above. Can be easily expanded.

  The molecular weight of the water-soluble non-peptide oligomer excluding the linker moiety is generally relatively low. Exemplary values for the molecular weight of the water soluble polymer include less than about 1500 daltons, less than about 1450 daltons, less than about 1400 daltons, less than about 1350 daltons, less than about 1300 daltons, less than about 1250 daltons, less than about 1200 daltons, Less than 1150 daltons, less than about 1100 daltons, less than about 1050 daltons, less than about 1000 daltons, less than about 950 daltons, less than about 900 daltons, less than about 850 daltons, less than about 800 daltons, less than about 750 daltons, less than about 700 daltons, about Less than 650 daltons, less than about 600 daltons, less than about 550 daltons, less than about 500 daltons, less than about 450 daltons, less than about 400 daltons, less than about 350 daltons, less than about 300 daltons, less than about 250 daltons, less than about 200 daltons, and About 10 Less than Dalton, and the like.

  Exemplary ranges of molecular weights of water-soluble non-peptide oligomers (other than the linker) include about 100 to about 1400 daltons, about 100 to about 1200 daltons, about 100 to about 800 daltons, about 100 to about 500 daltons, about 100 to Examples include about 400 daltons, about 200 to about 500 daltons, about 200 to about 400 daltons, about 75 to 1000 daltons, and about 75 to about 750 daltons.

The number of monomers in the water-soluble non-peptide oligomer is between about 1 and about 30 (including 1 and 30), between about 1 and about 25, between about 1 and about 20, between about 1 and about 15. , Preferably between about 1 and about 12, and between about 1 and about 10. In some cases, the number of consecutive monomers in the oligomer (and corresponding conjugate) is one of one, two, three, four, five, six, seven, or eight. One. In further embodiments, the oligomer (and corresponding conjugate) is 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 Containing monomers. In further embodiments, the oligomer (and corresponding conjugate) comprises 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 monomers in succession. Have. Thus, for example, when the water-soluble non-peptide oligomer contains CH ₃ — (OCH ₂ CH ₂ ) _n —, “n” is replaced with 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 , 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30, and about Within one or more ranges of between 1 and about 25, between about 1 and about 20, between about 1 and about 15, between about 1 and about 12, and between about 1 and about 10. An integer that can be

  When the water-soluble non-peptide oligomer has 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 monomers, these values are about Corresponds to methoxy end-capped oligos (ethylene oxide) having molecular weights of 75, 119, 163, 207, 251, 295, 339, 383, 427, and 471 daltons. When the oligomer has 11, 12, 13, 14, or 15 monomers, these values are methoxy with molecular weights corresponding to about 515, 559, 603, 647, and 691 daltons, respectively. Corresponds to oligo (ethylene oxide) end-capped with

  When a water-soluble non-peptide oligomer is bound to an opioid agonist (as opposed to stepwise adding one or more monomers to effectively “grow” the oligomer onto the opioid agonist) It is preferred that the composition comprising the active form of the non-peptide oligomer is monodispersed. However, in those cases, when a bimodal composition is used, the composition will have a bimodal distribution centered on any two of the above numbers of monomers. . Ideally, the polydispersity index, Mw / Mn, of each peak in the bimodal distribution is 1.01 or less, more preferably 1.001 or less, and more preferably 1.0005 or less. Most preferably, each peak has a MW / Mn value of 1.0000. For example, bimodal oligomers are 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, etc. 2-3, 2 -4, 2-5, 2-6, 2-7, 2-8, 2-9, 2-10, etc. 3-4, 3-5, 3-6, 3-7, 3-8, 3- 9, 3-10 etc., 4-5, 4-6, 4-7, 4-8, 4-9, 4-10 etc., 5-6, 5-7, 5-8, 5-9, 5- Exemplary monomeric subunits such as 10 etc., 6-7, 6-8, 6-9, 6-10 etc., 7-8, 7-9, 7-10 etc., and 8-9, 8-10 etc. Any of the possible combinations are possible.

  In some cases, a composition comprising an active form of a water-soluble non-peptide oligomer is also trimodal or tetramodal and has a range of monomer units as described above. Oligomer compositions with a well-defined mixture of oligomers (ie bimodal, trimodal, tetramodal, etc.) are oligomers of the desired profile (a mixture of two oligomers that differ only in the number of monomers is bimodal) A mixture of three oligomers that differ only in the number of monomers is trimodal, and a mixture of four oligomers that differ only in the number of monomers is tetramodal) Column chromatography of polydisperse oligomers can be obtained by mixing oligomers, or alternatively by restoring a “center cut” so as to obtain a mixture of oligomers in the desired defined molecular weight range Can be obtained from

  The water-soluble non-peptide oligomer is preferably obtained from a composition that is preferably unimolecular or monodisperse. That is, the oligomers in the composition have the same individual molecular weight value, not a molecular weight distribution. Some monodisperse oligomers can be purchased from commercial sources such as those available from Sigma-Aldrich, or alternatively prepared directly from commercially available starting materials such as Sigma-Aldrich. Can do. Water-soluble non-peptide oligomers are described in Chen Y. et al. Baker, G .; L. J. et al. Org. Chem. 6870-6873 (1999), International Patent No. WO 02/098949, and US Patent Application Publication No. 2005/0136031.

When present, the spacer moiety (the water-soluble non-peptide polymer binds to the opioid agonist through the spacer moiety) is a single bond, a single atom, such as an oxygen atom or a sulfur atom, two atoms, or multiple atoms. obtain. The spacer portion is typically but not necessarily straight. The spacer portion “X” is preferably hydrolytically stable and preferably enzymatically stable. Preferably, the spacer moiety “X” has an atomic chain length of less than about 12, preferably less than about 10, more preferably less than about 8, more preferably less than about 5. It has an atomic chain length, where length means the number of atoms in a single chain, not counting substituents. For example, a urea bond such as this R _oligomer- NH— (C═O) —NH—R ′ _drug is considered to have a chain length of three atoms ( —N H— C (O) —N H—). . In selected embodiments, the spacer moiety linkage does not include an additional spacer group.

  In some cases, the spacer moiety “X” comprises an ether, amide, urethane, amine, thioether, urea, or carbon-carbon bond. The functional groups discussed below and shown in the examples are typically used for bond formation. The spacer moiety may also comprise a spacer group (or be adjacent or located on the side), although less preferred, as described further below.

More specifically, in selected embodiments, the spacer moiety, X, is a “-” (ie, a small molecule opioid agonist that can be stable or degradable and a residue of a water-soluble non-peptide oligomer. Covalent bond between), —C (O) O—, —OC (O) —, —CH ₂ —C (O) O—, —CH ₂ —OC (O) —, —C (O) O—CH. _{2 -, - OC (O)} -CH 2, -O -, - NH -, - S -, - C (O) -, C (O) -NH, NH-C (O) -NH, OC _{(O) -NH, -C (S} ) -, - CH 2 -, - CH 2 -CH 2 -, - CH 2 -CH 2 -CH 2 -, - CH 2 -CH 2 -CH 2 -CH 2 - _{, -O-CH 2 -, -} CH 2 -O -, - O-CH 2 -CH 2 -, - CH 2 -O-CH 2 -, - CH 2 -CH 2 -O-, _{O-CH 2 -CH 2 -CH 2} -, - CH 2 -O-CH 2 -CH 2 -, - CH 2 -CH 2 -O-CH 2 -, - CH 2 -CH 2 -CH 2 -O- _{, -O-CH 2 -CH 2 -CH} 2 -CH 2 -, - CH 2 -O-CH 2 -CH 2 -CH 2 -, - CH 2 -CH 2 -O-CH 2 -CH 2 -, - _{CH 2 -CH 2 -CH 2 -O-} CH 2 -, - CH 2 -CH 2 -CH 2 -CH 2 -O -, - C (O) -NH-CH 2 -, - C (O) -NH _{-CH 2 -CH 2 -, - CH} 2 -C (O) -NH-CH 2 -, - CH 2 -CH 2 -C (O) -NH -, - C (O) -NH-CH 2 -CH _{2 -CH 2 -, - CH 2} -C (O) -NH-CH 2 -CH 2 -, - CH 2 -CH 2 -C (O) -NH-CH 2 -, - CH 2 _{-CH 2 -CH 2 -C (O)} -NH -, - C (O) -NH-CH 2 -CH 2 -CH 2 -CH 2 -, - CH 2 -C (O) -NH-CH 2 - _{CH 2 -CH 2 -, - CH} 2 -CH 2 -C (O) -NH-CH 2 -CH 2 -, - CH 2 -CH 2 -CH 2 -C (O) -NH-CH 2 -, - _{CH 2 -CH 2 -CH 2 -C (} O) -NH-CH 2 -CH 2 -, - CH 2 -CH 2 -CH 2 -CH 2 -C (O) -NH -, - NH-C (O _{) -CH 2 -, - CH 2} -NH-C (O) -CH 2 -, - CH 2 -CH 2 -NH-C (O) -CH 2 -, - NH-C (O) -CH 2 - _{CH 2 -, - CH 2 -NH} -C (O) -CH 2 -CH 2, -CH 2 -CH 2 -NH-C (O) -CH 2 -CH 2, -C (O) -NH- _{H 2 -, - C (O} ) -NH-CH 2 -CH 2 -, - O-C (O) -NH-CH 2 -, - O-C (O) -NH-CH 2 -CH 2 -, _{-NH-CH 2 -, - NH} -CH 2 -CH 2 -, - CH 2 -NH-CH 2 -, - CH 2 -CH 2 -NH-CH 2 -, - C (O) -CH 2 -, _{-C (O) -CH 2 -CH 2} -, - CH 2 -C (O) -CH 2 -, - CH 2 -CH 2 -C (O) -CH 2 -, - CH 2 -CH 2 -C _{(O) -CH 2 -CH 2 -} , - CH 2 -CH 2 -C (O) -, - CH 2 -CH 2 -CH 2 -C (O) -NH-CH 2 -CH 2 -NH-, _{-CH 2 -CH 2 -CH 2 -C (} O) -NH-CH 2 -CH 2 -NH-C (O) -, - CH 2 -CH 2 -CH 2 -C (O) -NH-CH 2 -C _{2 -NH-C (O) -CH} 2 -, a divalent cycloalkyl group, ^{R 6} is, H or alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, and from the group consisting of substituted aryl It can be any one of —N (R ⁶ ) — which is an organic radical selected.

  However, for the purposes of the present invention, a group of atoms is directly adjacent to the oligomer segment, and the group of atoms is the same as the oligomeric monomer so that the group simply represents an extension of the oligomer chain. In some cases, it is not considered a spacer.

  The bond “X” between the water-soluble non-peptide oligomer and the small molecule typically has one functional group on the end of the oligomer (or one when it is desired to “grow” the oligomer onto the opioid agonist). The above monomer) and the corresponding functional group of the opioid agonist are formed. The exemplary reaction is briefly described below. For example, the amino group on the oligomer may be reacted with a carboxylic acid or active carboxylic acid derivative on the small molecule, or vice versa, to produce an amide bond. Alternatively, an amine on the oligomer and an activated acid salt on the drug (eg, succinimidyl or benzotriazyl carbonate), or vice versa, forms a carbamate bond. Reactions of amines on oligomers with isocyanates on drugs (R—N═C═O), or vice versa, form urea linkages (R—NH (C═O) —NH—R ′). Furthermore, the reaction of alcohol (alkoxide) groups on the oligomer with alkyl halides within the drug, or halogenated groups, or vice versa, forms an ether linkage. In yet another coupling approach, small molecules with aldehyde functional groups are coupled to oligomeric amino groups by reductive amination, resulting in the formation of secondary amine bonds between the oligomer and the small molecule.

Particularly preferred water-soluble non-peptide oligomers are those bearing aldehyde functional groups. In this regard, the oligomer has the following structure: _{CH 3 O- (CH 2 -CH 2} -O) n - (CH 2) p -C (O) H, wherein, (n) is 1,2,3,4,5,6,7,8 , 9 and 10 and (p) is one of 1, 2, 3, 4, 5, 6 and 7. Preferred values for (n) include 3, 5, and 7, and preferred values for (p) are 2, 3, and 4. In addition, the carbon atom α for the —C (O) H moiety can be optionally substituted with alkyl.

  Typically, the end of a water-soluble non-peptide oligomer that does not carry a functional group is sealed to render it non-reactive. When the oligomer contains an additional functional group at the end, other than for the purpose of conjugation, that group is inactive under the formation conditions of the bond “X” or the bond “X” is formed. Selected to be protected inside.

As mentioned above, the water-soluble non-peptide oligomer contains at least one functional group prior to conjugation. Functional groups generally include electrophilic or nucleophilic groups for covalent attachment to small molecules based on reactive groups contained within or introduced into the small molecule. Examples of nucleophilic groups that may be present in the oligomer or small molecule include hydroxyl, amine, hydrazine (—NHNH ₂ ), hydrazide (—C (O) NHNH ₂ ), and thiol. Suitable nucleophiles include amines, hydrazines, hydrazides, and thiols, especially amines. Most small molecule drugs for covalent attachment to oligomers have a free hydroxyl, amino, thio, aldehyde, ketone, or carboxyl group.

  Examples of electrophilic functional groups that may be present in the oligomer or small molecule include carboxylic acids, carboxylic esters, especially imide esters, orthoesters, carbonates, isocyanates, isothiocyanates, aldehydes, ketones, thiones, alkenyls, acrylates, methacrylates. , Acrylamide, sulfone, maleimide, disulfide, iodo, epoxy, sulfonate, thiosulfonate, silane, alkoxysilane, and halosilane. More specific examples of these groups include succinimidyl ester or carbonate, imidazolyl ester or carbonate, benzotriazole ester or carbonate, vinyl sulfone, chloroethyl sulfone, vinyl pyridine, pyridyl disulfide, iodoacetamide, glyoxal, dione , Mesylate, tosylate, and tresylate (2,2,2-trifluoroethanesulfonate).

  Also, some sulfur analogs of these groups such as thione, thione hydrate, thioketal, 2-thiazolidinethione, etc., as well as hydrates or protected derivatives of any of the above moieties (eg, aldehydes) Hydrates, hemiacetals, acetals, ketone hydrates, hemiketals, ketals, thioketals, thioacetals).

An “active derivative” of a carboxylic acid generally refers to a carboxylic acid derivative that reacts readily with nucleophilic atoms much more easily than a non-derivatized carboxylic acid. Active carboxylic acids include, for example, acidic halides (acid chlorides, etc.), anhydrides, carbonates, and esters. Such esters include imide esters having the general form-(CO) ON-[[CO)-] ₂ , such as N-hydroxysuccinimidyl (NHS) ester or N-hydroxyphthalimidide. Luester. Also preferred are imidazolyl esters and benzotriazole esters. Particular preference is given to active propionic acid or butanoic acid esters as described in co-owned US Pat. No. 5,672,662. _, - _(CH 2) comprises 2-3 C (= O) O- Q in the form of a group, Q is, N- succinimide, N- sulfosuccinimide, N- phthalimide, N- glutarimide, N- tetrahydro It is preferably selected from phthalimide, N-norbornene-2,3-dicarboximide, benzotriazole, 7-azabenzotriazole, and imidazole.

  Other suitable electrophilic groups include succinimidyl carbonate, maleimide, benzotriazole carbonate, glycidyl ether, imidazolyl carbonate, p-nitrophenyl carbonate, acrylate, tresylate, aldehyde, and orthopyridyl disulfide. Can be mentioned.

  These electrophilic groups undergo a reaction with a nucleophilic atom such as a hydroxy, thio, or amino group to generate various bond types. In the present invention, a reaction that easily forms a hydrolytically stable bond is preferable. For example, carboxylic acids and their active derivatives, including ortho esters, succinimidyl esters, imidazolyl esters, and benzotriazole esters, react with nucleophilic atoms of the type described above to form esters, thioesters, and amides, respectively. Of these, amides are the most hydrolytically stable. Carbonates, including succinimidyl, imidazolyl, and benzotriazole carbonate, react with amino groups to form carbamates. Isocyanates (R—N═C═O) react with hydroxyl or amino groups to form carbamate (RNH—C (O) —OR ′) or urea (RNH—C (O) —NHR ′) bonds, respectively. Form. Aldehydes, ketones, glyoxals, diones, and their hydrates or alcohol adducts (ie, aldehyde hydrates, hemiacetals, acetals, ketone hydrates, hemiketals, and ketals) preferably react with amines. Subsequent reduction of the resulting imine, optionally providing an amine linkage (reductive amination).

  Some of the electrophilic functional groups contain electrophilic double bonds to which nucleophilic groups such as thiols can be added, for example to form thioether bonds. These groups include maleimide, vinyl sulfone, vinyl pyridine, acrylate, methacrylate, and acrylamide. Other groups include leaving groups that can be replaced by nucleophilic atoms, including chloroethyl sulfone, pyridyl disulfide (including cleavable SS bonds), iodoacetamide, mesylate, tosylate, thiosulfonate , And tresylate. Epoxides react by ring opening by nucleophilic atoms to form, for example, ether or amine bonds. The conjugates of the invention are prepared utilizing reactions involving complementary reactive groups as described above on oligomers and small molecules.

  In some cases, the opioid agonist may not have a functional group suitable for conjugation. In this case, it is possible to modify the “original” opioid agonist to have the desired functional group. For example, if the opioid agonist has an amide group but an amine group is desired, the Hofmann rearrangement, the Curtius rearrangement (amide is converted to azide once), or the Lossen rearrangement (amide is once converted to hydroxyamide, Subsequent treatment with toluene-2-sulfonyl chloride / base can be followed to modify the amide group to an amine group.

  A small molecule in which a small molecule opioid agonist carrying a carboxyl group carries a carboxyl group attached to an amino-terminal oligomer ethylene glycol so as to provide a conjugate with an amide group that covalently attaches the small molecule opioid agonist to the oligomer It is possible to prepare conjugates of opioid agonists. This is done, for example, by conjugating a small molecule opioid agonist bearing a carboxyl group with an amino-terminal oligomeric ethylene glycol in an anhydrous organic solvent in the presence of a coupling reagent (dicyclohexylcarbodiimide, or “DCC”). be able to.

  In addition, conjugates of small molecule opioid agonists in which the small molecule opioid agonist carrying a hydroxyl group carries a hydroxyl group attached to oligomer ethylene glycol so as to provide a small molecule conjugate of an ether (-O-) linkage. Can be prepared. This can be done, for example, by deprotonating the hydroxyl group using sodium hydride followed by reaction with an oligomeric ethylene glycol terminated with a halide.

  In other examples, conjugates of small molecule opioid agonists carrying a ketone group can be prepared by first reducing the ketone group to form the corresponding hydroxyl group. Thereafter, small molecule opioid agonists that now carry a hydroxyl group can be conjugated as described herein.

In yet another instance, conjugates of small molecule opioid agonists carrying amine groups can be prepared. In one approach, a small molecule opioid agonist bearing an amine group and an oligomer bearing an aldehyde are dissolved in a suitable buffer followed by the addition of a suitable reducing agent (eg, NaCNBH ₃ ). Subsequent to the reduction, as a result, an amine bond is formed between the amine group of the amine group-containing small molecule opioid agonist and the carbonyl carbon of the oligomer carrying the aldehyde.

  In another approach for preparing conjugates of small molecule opioid agonists carrying an amine group, an oligomer carrying a carboxylic acid and a small molecule opioid agonist carrying an amine group are typically combined with a binding reagent ( For example, the binding is performed in the presence of DCC). As a result, an amide bond is formed between the amine group of the amine group-containing small molecule opioid agonist and the carbonyl of the oligomer carrying the carboxylic acid.

式中、Ｒ^２、Ｒ^３、Ｒ^４、点線（「−−−」）、Ｙ^１、およびＲ^５のそれぞれは、化学式Ｉに関して定義された通りであり、Ｘは、スペーサー部分であり、ＰＯＬＹは、水溶性非ペプチドオリゴマーである。 Exemplary conjugates of the opioid agonist of Formula I include those having the following structure:

Wherein each of R ² , R ³ , R ⁴ , dotted line (“---”), Y ¹ , and R ⁵ is as defined with respect to Formula I, X is a spacer moiety, and POLY Is a water-soluble non-peptide oligomer.

式中、Ｒ^１、Ｒ^２、Ｒ^３、Ｒ^４、点線（「−−−」）、およびＹ^１のそれぞれは、化学式Ｉに関して定義された通りであり、Ｘは、スペーサー部分であり、ＰＯＬＹは、水溶性非ペプチドオリゴマーである。 Additional exemplary conjugates of formula I opioid agonists include those having the following structure:

Wherein ^each of R ¹ , R ² , R ³ , R ⁴ , dotted line (“---”), and Y ¹ is as defined with respect to Formula I, X is a spacer moiety, and POLY Is a water-soluble non-peptide oligomer.

式中、Ｒ^１、Ｒ^２、Ｒ^３、Ｒ^４、Ｙ^１、およびＲ^５のそれぞれは、化学式Ｉに関して定義された通りであり、Ｘは、スペーサー部分であり、ＰＯＬＹは、水溶性非ペプチドオリゴマーである。 Further additional exemplary conjugates of the opioid agonists of formula I include those having the following structure:

Wherein ^each of R ¹ , R ² , R ³ , R ⁴ , Y ¹ , and R ⁵ is as defined for Formula I, X is a spacer moiety, POLY is a water-soluble non-peptide It is an oligomer.

式中、Ｒ^１、Ｒ^２、Ｒ^３、Ｒ^４、Ｙ^１、およびＲ^５のそれぞれは、化学式Ｉに関して定義された通りであり、Ｘは、スペーサー部分であり、ＰＯＬＹは、水溶性非ペプチドオリゴマーである。 Further exemplary conjugates of the opioid agonists of formula I include those having the following structure:

Wherein ^each of R ¹ , R ² , R ³ , R ⁴ , Y ¹ , and R ⁵ is as defined for Formula I, X is a spacer moiety, POLY is a water-soluble non-peptide It is an oligomer.

式中、Ｒ^１、Ｒ^３、Ｒ^４、点線（「−−−」）、Ｙ^１、およびＲ^５のそれぞれは、化学式Ｉに関して定義された通りであり、Ｘは、スペーサー部分であり、ＰＯＬＹは、水溶性非ペプチドオリゴマーである。 Additional exemplary conjugates of formula I opioid agonists include those having the following structure:

Wherein ^each of R ¹ , R ³ , R ⁴ , dotted line (“---”), Y ¹ , and R ⁵ is as defined with respect to Formula I, X is a spacer moiety, and POLY Is a water-soluble non-peptide oligomer.

式中、存在する場合、Ｒ^１、Ｒ^２、Ｒ^３、Ｒ^４、点線（「−−−」）、Ｙ^１、およびＲ^５のそれぞれは、化学式Ｉに関して定義された通りであり、変数「ｎ」は、１〜３０の整数である。 Further exemplary conjugates include those having the following chemical formula:

Wherein, when present, each of R ¹ , R ² , R ³ , R ⁴ , dotted line (“---”), Y ¹ , and R ⁵ is as defined for Formula I and the variable “ “n” is an integer of 1 to 30.

式中、
Ｎ^＊は、窒素であり、
Ａｒは、シクロヘキシル、フェニル、ハロフェニル、メトキシフェニル、アミノフェニル、ピリジル、フリル、およびチエニルから成る群より選択され、
Ａｌｋは、エチレンおよびプロピレンから成る群より選択され、
Ｒ_ＩＩは、低級アルキル、低級アルコキシ、ジメチルアミノ、シクロプロピル、１−ピロリジル、モルフォリノから成る群より選択され（好ましくは、エチルなどの低級アルキル）、
Ｒ_ＩＩ’は、水素、メチル、およびメトキシから成る群より選択され、
Ｒ_ＩＩ”は、水素および有機ラジカルから成る群より選択され（好ましくは低級アルキル）、
Ｘは、リンカーであり（例えば、共有結合「−」または１つ以上の原子）、かつ
ＰＯＬＹは、水溶性非ペプチドオリゴマーである。
式ＩＩ−Ｃａに関して、条件に応じてアミンのうちの１つまたは両方（しかしより典型的には、化学式ＩＩ−Ｃａにおいて星印で標識されたアミン（「Ｎ^＊」））をプロトン化することができる。 Exemplary conjugates of the opioid agonist of Formula II include those having the following structure:

Where
N ^* is nitrogen,
Ar is selected from the group consisting of cyclohexyl, phenyl, halophenyl, methoxyphenyl, aminophenyl, pyridyl, furyl, and thienyl;
Alk is selected from the group consisting of ethylene and propylene;
R _II is selected from the group consisting of lower alkyl, lower alkoxy, dimethylamino, cyclopropyl, 1-pyrrolidyl, morpholino (preferably lower alkyl such as ethyl),
R _II ′ is selected from the group consisting of hydrogen, methyl, and methoxy;
R _II ″ is selected from the group consisting of hydrogen and organic radicals (preferably lower alkyl);
X is a linker (eg, a covalent bond “-” or one or more atoms) and POLY is a water-soluble non-peptide oligomer.
With respect to Formula II-Ca, protonating one or both of the amines (but more typically amines labeled with an asterisk (“N ^* ”) in Formula II-Ca) depending on conditions Can do.

式中、
Ｎ^＊は、窒素であり、
Ａｒは、シクロヘキシル、フェニル、ハロフェニル、メトキシフェニル、アミノフェニル、ピリジル、フリル、およびチエニルから成る群より選択され、
Ａｌｋは、エチレンおよびプロピレンから成る群より選択され、
Ｒ_ＩＩは、低級アルキル、低級アルコキシ、ジメチルアミノ、シクロプロピル、１−ピロリジル、モルフォリノから成る群より選択され（好ましくはエチルなどの低級アルキル）、
Ｒ_ＩＩ’は、水素、メチル、およびメトキシから成る群より選択され、
Ｒ_ＩＩ”は、水素および有機ラジカルから成る群より選択され（好ましくは低級アルキル）、
Ｘは、リンカーであり（例えば、共有結合「−」または１つ以上の原子）、かつ
ＰＯＬＹは、水溶性非ペプチドオリゴマーである。
化学式ＩＩ−Ｃｂに関して、条件に応じて、アミンのうちの１つまたは両方（しかしより典型的には、化学式ＩＩ−Ｃｂにおいて星印で標識されたアミン（「Ｎ^＊」））をプロトン化することができる。 Additional exemplary conjugates of Formula II opioid agonists include those having the following structure:

Where
N ^* is nitrogen,
Ar is selected from the group consisting of cyclohexyl, phenyl, halophenyl, methoxyphenyl, aminophenyl, pyridyl, furyl, and thienyl;
Alk is selected from the group consisting of ethylene and propylene;
R _II is selected from the group consisting of lower alkyl, lower alkoxy, dimethylamino, cyclopropyl, 1-pyrrolidyl, morpholino (preferably lower alkyl such as ethyl),
R _II ′ is selected from the group consisting of hydrogen, methyl, and methoxy;
R _II ″ is selected from the group consisting of hydrogen and organic radicals (preferably lower alkyl);
X is a linker (eg, a covalent bond “-” or one or more atoms) and POLY is a water-soluble non-peptide oligomer.
With respect to Formula II-Cb, depending on the conditions, one or both of the amines (but more typically the amine labeled with an asterisk (“N ^* ”) in Formula II-Cb) is protonated. be able to.

式中、
Ｎ^＊は、窒素であり、
Ａｒは、シクロヘキシル、フェニル、ハロフェニル、メトキシフェニル、アミノフェニル、ピリジル、フリル、およびチエニルから成る群より選択され、
Ａｌｋは、エチレンおよびプロピレンから成る群より選択され、
Ｒ_ＩＩは、低級アルキル、低級アルコキシ、ジメチルアミノ、シクロプロピル、１−ピロリジル、モルフォリノから成る群より選択され（好ましくはエチルなどの低級アルキル）、
Ｒ_ＩＩ’は、水素、メチル、およびメトキシから成る群より選択され、
Ｒ_ＩＩ”は、水素および有機ラジカルから成る群より選択され（好ましくは低級アルキル）、
各Ｘは独立して、リンカーであり（例えば、共有結合「−」または１つ以上の原子）、かつ
各ＰＯＬＹは独立して、水溶性非ペプチドオリゴマーである。
化学式ＩＩ−Ｃｃに関して、条件に応じて、アミンのうちの１つまたは両方（しかしより典型的には、化学式ＩＩ−Ｃｃにおいて星印で標識されたアミン（「Ｎ^＊」））をプロトン化することができる。 Additional exemplary conjugates of Formula II opioid agonists include those having the following structure:

Where
N ^* is nitrogen,
Ar is selected from the group consisting of cyclohexyl, phenyl, halophenyl, methoxyphenyl, aminophenyl, pyridyl, furyl, and thienyl;
Alk is selected from the group consisting of ethylene and propylene;
R _II is selected from the group consisting of lower alkyl, lower alkoxy, dimethylamino, cyclopropyl, 1-pyrrolidyl, morpholino (preferably lower alkyl such as ethyl),
R _II ′ is selected from the group consisting of hydrogen, methyl, and methoxy;
R _II ″ is selected from the group consisting of hydrogen and organic radicals (preferably lower alkyl);
Each X is independently a linker (eg, a covalent bond “-” or one or more atoms), and each POLY is independently a water-soluble non-peptide oligomer.
With respect to Formula II-Cc, depending on the conditions, one or both of the amines (but more typically the amine labeled with an asterisk (“N ^* ”) in Formula II-Cc) is protonated. be able to.

式中、変数「ｎ」は、１〜３０の整数である。 Additional exemplary conjugates are encompassed by the following formula:

In the formula, the variable “n” is an integer of 1 to 30.

式中、上記抱合体のそれぞれに対して、Ｘは、リンカー（例えば、共有結合「−」または１つ以上の原子）であり、ＰＯＬＹは、水溶性非ペプチドオリゴマーである。 Additional conjugates include those provided below

Where for each of the conjugates X is a linker (eg, a covalent bond “-” or one or more atoms) and POLY is a water-soluble non-peptide oligomer.

式中、
Ｒ^１は、アシルであり、
Ｒ^２は、水素、ハロゲン、非置換アルキルおよびハロゲンによって置換されたアルキルから成る群より選択され、
Ｒ^３は、ハロゲンおよびアルコキシから成る群より選択され、
Ｒ^５は、ヒドロキシル、エステル、アルコキシ、およびアルコキシアルキルから成る群より選択され、
Ａ_１は、アルキレンであり、
Ｘは、リンカーであり、
ＰＯＬＹは、水溶性非ペプチドオリゴマーである。 Additional conjugates include those provided below,

Where
R ¹ is acyl;
R ² is selected from the group consisting of hydrogen, halogen, unsubstituted alkyl and alkyl substituted by halogen;
R ³ is selected from the group consisting of halogen and alkoxy;
R ⁵ is selected from the group consisting of hydroxyl, ester, alkoxy, and alkoxyalkyl;
A ₁ is alkylene,
X is a linker;
POLY is a water-soluble non-peptide oligomer.

  The conjugates of the invention may exhibit a reduced blood brain barrier crossing rate. Further, the conjugate maintains at least about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70% or more of the biological activity of the unmodified parent small molecule drug.

  Although it is believed that the full range of conjugates disclosed herein has been described, the optimally sized oligomer can be determined as follows.

  First, an oligomer obtained from a monodisperse or bimodal water-soluble oligomer is conjugated to a small molecule drug. The drug is preferably orally bioavailable and as such exhibits a non-negligible blood brain barrier crossing rate. The ability of the conjugate to cross the blood brain barrier is then determined using an appropriate model and compared to that of the unmodified parent drug. If the results are favorable, i.e. if, for example, the crossing speed is significantly reduced, the biological activity of the conjugate is further evaluated. The compounds according to the present invention preferably have a significant degree of biological activity relative to the parent drug, ie more than about 30% of the biological activity of the parent drug, more preferably more than about 50% of the biological activity of the parent drug. maintain.

  The above steps are repeated one or more times using oligomers of the same type of monomer but different numbers of subunits and the results are compared.

  The oral bioavailability is assessed for individual conjugates whose ability to cross the blood brain barrier is reduced compared to unconjugated small molecule drugs. Based on these results, i.e. based on comparison of oligomer conjugates of different sizes with a given small molecule at a given location or location within a small molecule The size of the most effective oligomer in providing conjugates with an optimal balance between bioavailability and biological activity can be determined. Because the oligomer is small, such screening is possible and the properties of the resulting conjugate can be effectively adjusted. Conjugates with a favorable balance between reduced biomembrane transversal rate, bioactivity, and oral bioavailability, using small changes in oligomer size, and experimental design techniques Can be identified effectively. In some cases, the conjugation of the oligomers described herein is effective to actually increase the bioavailability of the drug.

  For example, one of ordinary skill in the art would use routine experimentation to first prepare a series of oligomers with different weights and functional groups, then administer the conjugate to the patient and perform regular blood and / or urine Sampling can be used to obtain the required clearance profile to determine the optimal molecular size and binding for improved oral bioavailability. Once a series of clearance profiles is obtained for each conjugate tested, suitable conjugates can be identified.

  Animal models (rodents and dogs) can also be used to study oral drug transport. In addition, non-in vivo methods include rodent inverted enterectomies and Caco-2 cell monolayer tissue culture models. These models are useful in predicting the bioavailability of oral drugs.

Such compounds can be tested to determine whether an opioid antagonist, or a conjugate of an opioid antagonist and a water-soluble non-peptide polymer, has activity as a mu opioid receptor antagonist. For example, K _D (binding affinity) and B _max (receptor number) are reported in Malatynska et al. (1995) NeuroReport 6 : 613-616. Briefly, the human mu receptor can be expressed in Chinese hamster ovary cells by recombinant techniques. Radioligand [ ³ H] -diprenorphine (30-50 Ci / mmol) with a final ligand concentration of [0.3 nM] can be used. Naloxone is used as a nonspecific confirmation [3.0 nM], reference compound, and positive control. The reaction is carried out in 50 mM TRIS-HCl (pH 7.4) containing 5 mM MgCl ₂ for 150 minutes at 25 ° C. The reaction is terminated by rapid vacuum filtration onto a glass fiber filter. The radioactivity captured on the filter is measured and compared to a control value to confirm the interaction of the test compound with the cloned mu binding site.

Similar tests can be performed on kappa opioid receptor agonists. For example, Lahti et al. (1985) Eur. Jrnl. Pharmac. 109 : 281-284, Rothman et al. (1992) Peptides 13 : 977-987, Kinouchi et al. (1991) Eur. Jrnl. Pharmac. 207 : 135-141. Briefly, human kappa receptors can be obtained from the guinea pig cerebellar membrane. Radioligand [ ³ H] -U-69593 (40-60 Ci / mmol) with a final ligand concentration of [0.75 nM] can be used. U-69593 is used as non-specific confirmation [1.0 nM], reference compound and positive control. The reaction is carried out in 50 mM HEPES (pH 7.4) for 120 minutes at 30 ° C. The reaction is terminated by rapid vacuum filtration onto a glass fiber filter. The radioactivity captured on the filter is measured and compared to a control value to confirm the interaction of the test compound with the cloned kappa binding site.

  The conjugates described herein include not only the conjugate itself, but also conjugates in the form of pharmaceutically acceptable salts. The conjugates described herein can have a sufficiently acidic group, a sufficiently basic group, or both functional groups, and thus some inorganic bases, as well as inorganic and organic acid It can react with any of them to form a salt. The acids commonly employed to form acid addition salts are inorganic acids such as hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, phosphoric acid, and the like, p-toluenesulfonic acid, methanesulfonic acid Organic acids such as oxalic acid, p-bromophenyl-sulfonic acid, carbonic acid, succinic acid, citric acid, benzoic acid, acetic acid, and the like. Examples of such salts include sulfate, pyrosulfate, bisulfate, sulfite, phosphate, monohydrogen phosphate, dihydrogen phosphate, metaphosphate, pyrophosphate, chloride, Bromide, iodide, acetate, propionate, decanoate, caprylate, acrylate, formate, isobutyrate, caproate, heptanoate, propiolate, oxalate, malonate , Succinate, suberate, sebacate, fumarate, maleate, butyne-1,4-dioate, hexyne-1,6-dioate, benzoate, chlorobenzoate, methylbenzoate , Dinitrobenzoate, hydroxybenzoate, methoxybenzoate, phthalate, sulfonate, xylenesulfonate, phenylacetate, phenylpropionate, phenylbutyrate, citrate, lactic acid , Γ-hydroxybutyrate, glycolate, tartrate, methanesulfonate, propanesulfonate, naphthalene-1-sulfonate, naphthalene-2-sulfonate, mandelate, and the like Including.

  Base addition salts include those derived from inorganic salts such as ammonium hydroxide or alkali metal hydroxides or alkaline earth metal carbonates, carbonates, bicarbonates, and the like. Such salts useful in preparing the salts of the present invention thus include sodium hydroxide, potassium hydroxide, ammonium hydroxide, potassium carbonate, and the like.

  The present invention also includes a pharmaceutical comprising a conjugate provided herein in combination with a pharmaceutical excipient. In general, the conjugate itself is in a solid form (eg, a precipitate) and can be combined with a suitable pharmaceutical excipient that can be in either a solid or liquid form.

  Exemplary excipients include those selected from the group consisting of carbohydrates, inorganic salts, antibacterial agents, antioxidants, surfactants, buffers, acids, bases and combinations thereof. It is not limited.

  Carbohydrates such as sugars, derivatized sugars such as alditols, aldonic acids, esterified sugars, and / or sugar polymers may be present as excipients. Specific carbohydrate excipients include, for example, monosaccharides such as fructose, maltose, galactose, glucose, D-mannose, sorbose, disaccharides such as lactose, sucrose, trehalose, cellobiose, raffinose, meretitol, maltodextrin, dextran And polysaccharides such as starch, and alditols such as mannitol, xylitol, maltitol, lactitol, xylitol, sorbitol (glucitol) pyranosyl sorbitol, and myo-inositol.

  Excipients also include inorganic salts or buffers such as citric acid, sodium chloride, potassium chloride, sodium sulfate, potassium nitrate, monobasic sodium phosphate, dibasic sodium phosphate, and combinations thereof.

  The preparation may include an antimicrobial agent to prevent or inhibit microbial growth. Non-limiting examples of antimicrobial agents suitable for the present invention include benzalkonium chloride, benzethonium chloride, benzyl alcohol, cetylpyridinium chloride, chlorobutanol, phenol, phenylethyl alcohol, phenylmercuric nitrate, thimerosal, and combinations thereof Is mentioned.

  Antioxidants can also be present in the formulation. Antioxidants are used to prevent oxidation, thereby preventing degradation of the conjugate or other components of the formulation. Suitable antioxidants for use in the present invention include, for example, ascorbyl palmitate, butylated hydroxyanisole, butylated hydroxytoluene, hypophosphorous acid, monothioglycerol, propyl gallate, sodium bisulfite, sodium formaldehyde sulfoxylate , Sodium metabisulfite, and combinations thereof.

  A surfactant may be present as an excipient. Exemplary surfactants include polysorbates such as “Tween 20” and “Tween 80”, and pluronics such as F68 and F88 (both available from BASF, Mount Olive, New Jersey), sorbitan esters, lecithin And other phospholipids such as phosphatidylcholine, phosphatidylethanolamine (but preferably not in liposome form), lipids such as fatty acids and fatty acid esters, steroids such as cholesterol, and EDTA, zinc, and other such suitable And chelating agents such as cations.

  A pharmaceutically acceptable acid or base may be present as an excipient in the formulation. Non-limiting examples of acids that can be used include hydrochloric acid, acetic acid, phosphoric acid, citric acid, malic acid, lactic acid, formic acid, trichloroacetic acid, nitric acid, perchloric acid, phosphoric acid, sulfuric acid, fumaric acid, and combinations thereof One selected from the group consisting of: Examples of suitable bases include sodium hydroxide, sodium acetate, ammonium hydroxide, potassium hydroxide, ammonium acetate, potassium acetate, sodium phosphate, potassium phosphate, sodium citrate, sodium formate, sodium sulfate, potassium sulfate , A base selected from the group consisting of potassium fumarate, and combinations thereof, but is not limited thereto.

  The amount of conjugate in the composition will vary depending on a number of factors, but is optimally a therapeutically effective amount when the composition is stored in a unit dose container. A therapeutically effective amount can be determined empirically by increasing doses of conjugate and repeatedly administering to determine which amount achieves the clinically desired endpoint.

  The amount of any individual excipient in the composition will vary depending on the activity of the excipient and the particular needs of the composition. In general, the optimal amount of any individual excipient is determined through routine experimentation, i.e., preparing compositions containing various amounts of excipients (ranging from small to large), stability and It is determined by examining other parameters and then determining the range where optimal performance is obtained without causing any significant adverse effects.

  In general, however, excipients are shaped in an amount of about 1 to about 99% by weight, preferably about 5 to 98% by weight, more preferably about 15 to 95% by weight, most preferably less than 30% by weight. As an agent, present in the composition.

Along with other excipients, general teachings regarding these above-described pharmaceutical excipients and pharmaceutical compositions are described in “Remington: The Science & Practice of Pharmacy”, 19 ^th ed. , Williams & Williams (1995), “Physician's Desk Reference”, 52 ^nd ed. , Medical Economics, Montvale, NJ (1998), and Kibbe, A. et al. H. ^{, Pharmaceutical Excipients, 3 rd Edition,} American Pharmaceutical Association, Washington, D. C. , 2000.

  The pharmaceutical composition can take several forms, and the invention is not limited in this regard. Exemplary formulations include tablets, caplets, capsules, gelcaps, troches, dispersions, suspensions, solvents, elixirs, syrups, lozenges, transdermal patches, sprays, suppositories, And most preferably in a form suitable for oral administration, such as powders and the like.

  Oral dosage forms are suitable for orally effective conjugates and include tablets, caplets, capsules, gelcaps, suspensions, solvents, elixirs, and syrups, and optionally capsules. It may also contain a plurality of granulated granules, beads, powders or pellets. Such dosage forms are prepared using conventional methods known in the pharmaceutical formulation art and described in the relevant documents.

  Tablets and caplets can be manufactured, for example, using standard tablet processing procedures and equipment. Direct compression and granulation techniques are preferred when preparing tablets or caplets containing the conjugates described herein. In addition to conjugates, tablets and caplets generally contain inert pharmaceutically acceptable carrier materials such as binders, lubricants, disintegrants, fillers, stabilizers, surfactants, colorants, and the like. contains. The binder is used to impart tackiness to the tablet and thus keep the tablet intact. Suitable binder materials include starch (including corn starch and pregelatinized starch), gelatin, sugars (including sucrose, glucose, dextrose, and lactose), polyethylene glycols, waxes, and natural and synthetic rubbers such as , Sodium acaciaate, polyvinylpyrrolidone, cellulose polymers (including hydroxypropylcellulose, hydroxypropylmethylcellulose, methylcellulose, microcrystalline cellulose, ethylcellulose, hydroxyethylcellulose, etc.), and Veegum, but are not limited to these. Lubricants are used to facilitate tablet manufacture, promote powder flow and prevent particle crowning (ie, particle breakage) when pressure is reduced. Useful lubricants are magnesium stearate, calcium stearate, and stearic acid. Disintegrants are used to facilitate tablet disintegration and are generally starches, clays, celluloses, algins, gums or cross-linked polymers. Fillers include, for example, materials such as silicon dioxide, titanium dioxide, alumina, talc, kaolin, powdered cellulose, and microcrystalline cellulose, as well as mannitol, urea, sucrose, lactose, dextrose, sodium chloride, and sorbitol. Soluble substances are mentioned. Stabilizers are used, as is well known in the art, to inhibit or prevent drug degradation reactions, including oxidative reactions, as an example.

  Capsules are also suitable oral dosage forms, in which the conjugate-containing composition can be a liquid or gel (eg, in the case of a gel cap), or a solid (granule, bead, powder, or pellet, such as a pellet) Can be encapsulated. Suitable capsules include hard and soft capsules and are generally made of gelatin, starch, or cellulose materials. The two-piece hard gelatin capsule is preferably sealed with a gelatin band or the like.

  Substantially dry forms of parenteral formulations (generally as lyophilisates or precipitates, which can be in the form of powders or cakes), and generally liquid, dry forms of parenteral formulations Formulations prepared for injection that require a step of reconstitution. Examples of suitable diluents for reconstituting the solid composition prior to injection include bacteriostatic water for injection, 5% dextrose water, phosphate buffered saline, Ringer's solution, saline, sterile water, deionized Water, and combinations thereof.

  In some cases, compositions intended for parenteral administration can take the form of non-aqueous solutions, suspensions, or emulsions, each typically being sterile. Examples of non-aqueous solvents or vehicles include propylene glycol, polyethylene glycol, vegetable oils such as olive oil and corn oil, gelatin, and injectable organic esters such as ethyl oleate.

  The parenteral formulations described herein can also contain adjuvants such as preservatives, wetting agents, emulsifying agents, and dispersing agents. The formulation is sterilized by introduction of a sterilant, filtration through a filter that retains bacteria, irradiation, or heat.

  Conjugates can also be administered through the skin using conventional transdermal patches or other transdermal delivery systems, in which the conjugate functions as a drug delivery means secured to the skin. Contained in the laminated structure. In such a structure, the conjugate is contained in a layer underneath the top backing layer, or “reservoir”. The laminated structure can contain a single reservoir or it can contain multiple reservoirs.

  Conjugates can also be formulated in suppositories for rectal administration. For suppositories, conjugates are coca butter (cocoa butter), polyethylene glycol, glycerin-treated gelatin, fatty acids, and combinations thereof (eg, solid at room temperature but softened, melted or dissolved at body temperature). Form) mixed with suppository base material. Suppositories include, for example, melting a suppository base material to form a melt, introducing a conjugate (before or after melting the suppository base material), and injecting the lysate into a mold. Preparing by cooling the melt (eg, placing the melt-containing mold in a room temperature environment) thereby forming a suppository and removing the suppository from the mold. Yes (not necessarily in the order shown).

  The invention also provides a method for administering a conjugate provided herein to a patient suffering from a condition responsive to treatment with the conjugate. The method generally involves administering a therapeutically effective amount of a conjugate, preferably provided as part of a pharmaceutical, orally. Other modes of administration are also contemplated, such as pulmonary, nasal, buccal, rectal, sublingual, transdermal, parenteral. As used herein, the term “parenteral” includes subcutaneous, intravenous, intraarterial, intraperitoneal, intracardiac, intrathecal, and intramuscular injection, and infusion injection.

  When parenteral administration is utilized, the molecular weight is about 500-30 K Daltons (eg, molecular weights of about 500, 1000, 2000, 2500, 3000, 5000, 7500, 10,000, somewhat larger than those described above. , 15000, 20000, 25000, 30000, or larger) oligomers may be required.

  This method of administration can be used to treat any condition that can be cured or prevented by administration of a particular conjugate. Those skilled in the art are aware of which conditions specific conjugates can effectively treat. The actual dose to be administered will vary based on the age, weight, and general condition of the subject, as well as the severity of the condition being treated, the judgment of the medical professional, and the conjugate being administered. Therapeutically effective amounts are known to those skilled in the art and / or are described in related reference documents and literature. In general, a therapeutically effective amount is in the range of about 0.001 mg to 1000 mg, preferably 0.01 mg / day to 750 mg / day, more preferably 0.10 mg / day to 500 mg / day.

  The unit dosage of any given conjugate (also preferably provided as part of a pharmaceutical formulation) can be administered in various dosing schedules depending on the judgment of the clinician, patient requirements, etc. it can. Specific dosing schedules are known to those skilled in the art or can be determined experimentally using routine methods. Exemplary dosing schedules include 5 times per day, 4 times per day, 3 times per day, 2 times per day, 1 time per day, 3 times per week, 2 times per week, 1 time per week, 2 times per month, monthly Including, but not limited to, once and any combination thereof. Once the clinical endpoint is achieved, dosing of the composition is stopped.

  One advantage of administering a conjugate of the invention is that a reduction in first pass metabolism can be achieved compared to the parent drug. Such a result is advantageous for many orally administered drugs that are greatly metabolized by passing through the intestine. In this way, the clearance of the conjugate can be adjusted by selecting the size, binding, and covalent position of the oligomeric molecule that provides the desired clearance properties. One skilled in the art can determine the ideal molecular size of the oligomer based on the teachings herein. A suitable decrease in first-pass metabolism of the conjugate compared to the corresponding unconjugated small drug molecule is at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least About 60%, at least about 70%, at least about 80% and at least about 90%.

  Accordingly, the present invention provides a method for reducing the metabolism of an active agent. The method comprises providing a monodisperse or bimodal conjugate, each conjugate comprising a small molecule drug-derived moiety covalently linked to the water-soluble oligomer by a stable linkage, wherein the conjugate is , Exhibiting a decrease in metabolic rate compared to the metabolic rate of a small molecule drug that is not bound to a water-soluble oligomer, and administering the conjugate to a patient. In general, administration is via one dosage form selected from the group consisting of oral administration, transdermal administration, buccal administration, transmucosal administration, vaginal administration, rectal administration, parenteral administration, and pulmonary administration. Is called.

  While it is useful to be able to reduce many types of metabolism (including both phase 1 and phase 2 metabolism), the conjugates can be used to convert small molecule drugs into liver enzymes (eg, cytochrome P450 isoforms). Particularly useful when metabolized by one or more of the foams) and / or by one or more intestinal enzymes.

  All articles, books, patents, patent publications, and other publications cited herein are incorporated by reference in their entirety. In the event of a conflict between the teachings herein and the teachings of the field incorporated by reference, the meaning of the teachings herein shall prevail.

(Experiment)
Although the invention has been described with reference to certain preferred specific embodiments, the foregoing description and the following examples are intended to be illustrative and not limiting the scope of the invention. It should be understood that there is no. Other aspects, advantages, and modifications within the scope of the invention will be apparent to those skilled in the art to which the invention pertains.

  Unless otherwise specified, all chemical reagents cited in the appended examples are commercially available. The preparation of PEG-mer is described, for example, in US Patent Application Publication No. 2005/0136031.

All ¹ H NMR (nuclear magnetic resonance) data was obtained with an NMR spectrometer manufactured by Bruker (MHz ≧ 200). A list of specific compounds as well as compound sources is provided below.

Example 1
(Preparation of oligomer-nalbuphine conjugate- "Method A")
PEG-nalbuphine was prepared using the first procedure. Schematically, the technique applied to the present embodiment is shown below.

(Desalination of nalbuphine hydrochloride dihydrate)
Nalbuphine hydrochloride dihydrate (600 mg, manufactured by Sigma) was dissolved in water (100 mL). Saturated aqueous K ₂ CO ₃ was added and then the pH was adjusted to 9.3 with 1N HCl solution saturated with sodium chloride. The solution was extracted with dichloromethane (5 × 25 mL). The combined organic solution was washed with brine (100 mL), dried over Na ₂ SO ₄ , concentrated to dryness and dried under high vacuum to yield nalbuphine (483.4 mg, 97% recovery). The product was confirmed by ¹ H-NMR in CDCl ₃ .

(Synthesis of 3-O-mPEG ₃ -nalbuphine (2) (n = 3))

Nalbuphine (28.5 mg, 0.08 mmol) was dissolved in a mixture of acetone (2 mL) and toluene (1.5 mL). Potassium carbonate (21 mg, 0.15 mmol) was added, followed by mPEG ₃ -Br (44.5 mg, 0.20 mmol) at room temperature. The resulting mixture was stirred at room temperature for 27.5 hours. Further potassium carbonate (24 mg, 0.17 mmol) was added. The mixture was heated with CEM microwave to reach 60 ° C. in 20 minutes and then to 100 ° C. in 30 minutes. DMF (0.2 mL) was added. The mixture was heated in the microwave at 60 ° C. for 20 minutes and 100 ° C. for 30 minutes. The reaction was concentrated to remove organic solvent and the residue was mixed with water (10 mL) and extracted with dichloromethane (4 × 15 mL). The combined organic solution was washed with brine, dried over Na ₂ SO ₄ and concentrated. The crude product was confirmed by HPLC and LC-MS. The residue was re-mixed with water (10 mL), adjusted to pH 2.3 with 1N HCl and washed with dichloromethane (2 × 15 mL). The aqueous solution was adjusted to pH 10.4 with 0.2N NaOH and extracted with dichloromethane (4 × 15 mL). The combined organic solution was washed with brine, dried over Na ₂ SO ₄ and concentrated. The residue using 0-10% MeOH in dichloromethane and purified by Biotage flash column chromatography, in 81% yield, the desired product _3-O-mPEG 3 - nalbuphine (2) (n = 3 ) (32.7 mg) was obtained. The product was confirmed by ¹ H-NMR, LC-MS.

(Synthesis of 3-O-mPEG ₄ -Nalbuphine (2) (n = 4))

A mixture of nalbuphine (96 mg, 0.27 mmol) and mPEG ₄ -OMs (131 mg, 0.46 mmol) in acetone (8 mL) was heated to reflux for 16 hours in the presence of potassium carbonate (113 mg, 0.82 mmol). Cool to room temperature, filter, and wash the solid with acetone and DCM. The solution was collected and concentrated to dryness. The residue was purified by Biotage automated flash column chromatography using 0-10% MeOH in dichloromethane to yield the product 3-O-mPEG ₄ -nalbuphine (2) (n = 4) in 74% yield. (109 mg) was obtained. The product was confirmed by ¹ H-NMR, LC-MS.

(Synthesis of 3-O-mPEG ₅ -Nalbuphine (2) (n = 5))

A mixture of nalbuphine (78.3 mg, 0.22 mmol) and mPEG ₅ -OMs (118 mg, 0.36 mmol) in acetone (8 mL) was heated at reflux for 16 hours in the presence of potassium carbonate (93 mg, 0.67 mmol). And cooled to room temperature, filtered, and the solid was washed with acetone and DCM. The solution was collected and concentrated to dryness. The residue was purified by Biotage automated flash column chromatography using 0-10% MeOH in dichloromethane to yield product 3-O-mPEG ₅ -nalbuphine (2) (n = 5) in 76% yield. (101 mg) was obtained. The product was confirmed by ¹ H-NMR, LC-MS.

(Synthesis of 3-O-mPEG ₆ -Nalbuphine (2) (n = 6))

A mixture of nalbuphine (89.6 mg, 0.25 mmol) and mPEG ₆ -OMs (164 mg, 0.44 mmol) in acetone (8 mL) was heated at reflux for 18 hours in the presence of potassium carbonate (98 mg, 0.71 mmol). And cooled to room temperature, filtered, and the solid was washed with acetone and DCM. The solution was collected and concentrated to dryness. The residue was purified by Biotage automated flash column chromatography using 0-10% MeOH in dichloromethane to yield the product 3-O-mPEG ₆ -nalbuphine (2) (n = 6) in 91% yield. (144 mg) was obtained. The product was confirmed by ¹ H-NMR, LC-MS.

(Synthesis of 3-O-mPEG ₇ -Nalbuphine (2) (n = 7))

A mixture of nalbuphine (67 mg, 0.19 mmol) and mPEG ₇ -Br (131 mg, 0.33 mmol) in acetone (10 mL) was heated to reflux in the presence of potassium carbonate (67 mg, 0.49 mmol) for 6 hours. Cool to room temperature, filter and wash the solid with acetone and dichloromethane. The solution was concentrated to dryness. The residue was purified by Biotage automated flash column chromatography using 2-10% MeOH in dichloromethane to give the product 3-O-mPEG ₇ -nalbuphine (2) (n = 7) (40.6 mg). Obtained. The product was confirmed by ¹ H-NMR, LC-MS.

(Synthesis of 3-O-mPEG ₈ -Nalbuphine (2) (n = 8))

Mixture of nalbuphine (60 mg, 0.17 mmol) and mPEG ₈ -Br (105.7 mg, 0.24 mmol) in toluene / DMF (3 mL / 0.3 mL) in the presence of potassium carbonate (40.8 mg, 0.30 mmol) Under heating with CEM microwave to reach 100 ° C. in 30 minutes. Acetone (1 mL) was then added. The mixture was heated with CEM microwave to reach 100 ° C. in 90 minutes, then additional K ₂ CO ₃ (31 mg, 0.22 mmol) and mPEG ₈ -Br (100 mg, 0.22 mmol) were added. The mixture was heated with CEM microwave to reach 100 ° C. in 60 minutes. mPEG ₈ -Br (95 mg, 0.21 mmol) was added again. The mixture was heated again with CEM microwave to reach 100 ° C. in 30 minutes. The reaction mixture was concentrated under reduced pressure. The residue was mixed with water (2 mL) and brine (10 mL). The pH of the solution was adjusted to 1.56 with 1N HCl and extracted with dichloromethane (3 × 20 mL). The combined organic solution was dried over Na ₂ SO ₄ and concentrated to yield residue I (mixture of desired product and precursor material). The aqueous solution was changed to pH 10.13 with 0.2N NaOH and extracted with dichloromethane (4 × 15 mL). The organic solution was washed with brine, dried over Na ₂ SO ₄ and concentrated to give residue II (19.4 mg) containing the product and starting material nalbuphine. Residue I was purified by Biotage automated flash column chromatography using 2-10% MeOH in dichloromethane to give the product 3-O-mPEG ₈ -nalbuphine (2) (n = 8) (44.6 mg). Obtained. The product was confirmed by ¹ H-NMR, LC-MS.

(Example 2)
(Preparation of oligomer-nalbuphine conjugate- "Method B"
PEG-nalbuphine was prepared using the second approach. Schematically, the technique applied to the present embodiment is shown below.

(Synthesis of 3-O-MEM-Nalbuphine (3))

Nalbuphine (321.9 mg, 0.9 mmol) was dissolved in acetone / toluene (19 mL / 8 mL). Then potassium carbonate (338 mg, 2.45 mmol) was added followed by MEMCl (160 μL, 1.41 mmol). The resulting mixture was stirred at room temperature for 21 hours. MeOH (0.3 mL) was added to quench the reaction. The reaction mixture was concentrated under reduced pressure and dried. The residue was mixed with water (5 mL) and brine (15 mL) and extracted with dichloromethane (3 × 15 mL). The combined organic solution was washed with brine, dried over Na ₂ SO ₄ and concentrated. The residue was separated by Biotage automated flash column chromatography using 2-10% MeOH in dichloromethane to give product 3-O-MEM-nalbuphine (3) (341 mg) and starting nalbuphine (19.3 mg). Got. The product was confirmed by ¹ H-NMR, LC-MS.

(Synthesis of _{6-O-mPEG 3 -3-} O-MEM- nalbuphine (4) (n = 3) )

A 20 mL vial was charged with 3-O-MEM-nalbuphine (3) (85 mg, 0.19 mmol) and toluene (15 mL). The mixture was concentrated to remove 7 mL of toluene. Anhydrous DMF (0.2 mL) was added. The vial was washed with nitrogen. NaH (60% dispersion in mineral oil, 21 mg, 0.53 mmol) was added, followed by mPEG ₃ -OMs (94 mg, 0.39 mmol). The resulting mixture was heated at 45 ° C. for 22.5 hours before additional NaH (22 mg, 0.55 mmol) was added. The mixture was heated at 45 ° C. for a further 6 hours, NaH (24 mg) was added and the mixture was heated at 45 ° C. for a further 19 hours. When the mixture was cooled to room temperature, saturated aqueous NaCl (1 mL) was added to quench the reaction. The mixture was diluted with water (10 mL) and extracted with EtOAc (4 × 15 mL). The combined organic solution was washed with brine, dried over Na ₂ SO ₄ and concentrated. The residue using 0-10% MeOH in dichloromethane, and separated by Biotage automated flash column chromatography, in 71% yield, product _{6-O-mPEG 3 -3-} O-MEM- nalbuphine (4 ) (N = 3) (79.4 mg) was obtained. The product was confirmed by ¹ H-NMR, LC-MS.

(Synthesis of 6-O-mPEG ₃ -Nalbuphine (5) (n = 3))

The _{6-O-mPEG 3 -3-} O-MEM- nalbuphine (4) (79.4mg), 6 hours at room temperature, was stirred in 2M HCl in methanol. The mixture was diluted with water (5 mL) and concentrated to remove methanol. The aqueous solution was washed with dichloromethane (5 mL) and the pH of the solution was adjusted to 9.35 with 0.2 N NaOH and solid NaHCO ₃ and extracted with dichloromethane (4 × 30 mL). The combined organic solution was washed with brine, dried over Na ₂ SO ₄ , concentrated and the product 6-O-mPEG ₃ -nalbuphine (5) (n = 3) (62. 5 mg) was obtained. The product was confirmed by ¹ H-NMR, LC-MS.

(Synthesis of _{6-O-mPEG 4 -3-} O-MEM- nalbuphine (4) (n = 4) )

A 50 mL round bottom flask was charged with 3-O-MEM-nalbuphine (3) (133.8 mg, 0.3 mmol), mPEG ₄ -OMs (145 mg, 0.51 mmol), and toluene (20 mL). The mixture was concentrated to remove about 12 mL of toluene. Anhydrous DMF (0.2 mL) was added. NaH (60% dispersion in mineral oil, 61 mg, 1.52 mmol) was added. The resulting mixture was heated at 45 ° C. for 21.5 hours before additional NaH (30 mg, 0.75 mmol) was added. The mixture was heated at 45 ° C. for an additional 5 hours. When the mixture was cooled to room temperature, saturated aqueous NaCl (1 mL) was added to quench the reaction. The mixture was diluted with water (15 mL) and extracted with EtOAc (4 × 15 mL). The combined organic solution was washed with brine, dried over Na ₂ SO ₄ and concentrated. The residue using 0-10% MeOH in dichloromethane and purified by Biotage automated flash column chromatography on silica gel, the product _{6-O-mPEG 4 -3-} O-MEM- nalbuphine (4) (n = 4) (214.4 mg) was obtained. ¹ H-NMR showed some mPEG ₄ -Oms in the product. No further purification was attempted. The product was confirmed by ¹ H-NMR, LC-MS.

(Synthesis of 6-O-mPEG ₄ -Nalbuphine (5) (n = 4))

The _{6-O-mPEG 4 -3-} O-MEM- nalbuphine (4) (214.4mg), 6 hours at room temperature, was stirred in 2M HCl in methanol (30 mL). The mixture was diluted with water (5 mL) and concentrated to remove methanol. The aqueous solution was adjusted to pH 9.17 with 1N NaOH and extracted with dichloromethane (4 × 25 mL). The combined organic solution was washed with brine, dried over Na ₂ SO ₄ and concentrated. The residue was purified by flash column chromatography on silica gel using 3-8% MeOH / DCM (Biotage) to give pure product 6-O-mPEG ₄ − with some impure product. Nalbuphine (5) (n = 4) (90.7 mg) was obtained. The product was confirmed by ¹ H-NMR, LC-MS. The impure part was dissolved in DCM (about 1.5 mL). 1N HCl in ether (20 mL) was added and centrifuged. The residue was collected and redissolved in DCM (25 mL). The DCM solution was washed with 5% aqueous NaHCO ₃ (20 mL), brine (2 × 30 mL), dried over Na ₂ SO ₄ and concentrated to give another pure product fraction (24.8 mg). It was.

(Synthesis of 6-O-mPEG ₅ -3-O-MEM-nalbuphine (4) (n = 5))

A 50 mL round bottom flask was charged with 3-O-MEM-nalbuphine (3) (103.9 mg, 0.23 mmol), mPEG ₅ -OMs (151 mg, 0.46 mmol), and toluene (38 mL). The mixture was concentrated to remove about 20 mL of toluene. Anhydrous DMF (0.5 mL) was added. NaH (60% dispersion in mineral oil, 102 mg, 2.55 mmol) was added. The resulting mixture was heated at 45 ° C. for 18 hours before additional NaH (105 mg) was added. The mixture was heated at 45 ° C. for an additional 5.5 hours. NaH (87 mg) was added and the mixture was heated at 45 ° C. for an additional 17.5 hours. When the mixture was cooled to room temperature, saturated aqueous NaCl (3 mL) was added to quench the reaction. The mixture was diluted with water (10 mL) and extracted with EtOAc (4 × 20 mL). The combined organic solution was washed with brine, dried over Na ₂ SO ₄ and concentrated. The residue using 3-8% MeOH in dichloromethane, and separated by Biotage automated flash column chromatography on silica gel, the product _{6-O-mPEG 5 -3-} O-MEM- nalbuphine (4) (n = 5) was obtained.

(Synthesis of 6-O-mPEG ₅ -Nalbuphine (5) (n = 5))

The above _{6-O-mPEG 5 -3-} O-MEM- nalbuphine (4), at room temperature for 2.5 h, was stirred in 2M HCl in methanol (30 mL). The mixture was diluted with water (5 mL) and concentrated to remove methanol. The aqueous solution was adjusted to pH 9.19 with 1N NaOH and extracted with dichloromethane (4 × 15 mL). The combined organic solution was washed with brine, dried over Na ₂ SO ₄ and concentrated. After purification by flash column chromatography on silica, mPEG ₅ -Oms was observed in ¹ H-NMR. The residue was dissolved in DCM (about 1 mL). 1N HCl in ether (18 mL) was added and centrifuged. The residue was collected and redissolved in DCM (25 mL). The DCM solution was washed with 5% aqueous NaHCO ₃ (2 × 20 mL), brine (2 × 30 mL), dried over Na ₂ SO ₄ and concentrated. The residue was purified by Biotage automated flash column chromatography on silica gel using 4-8% MeOH in dichloromethane to give the product 6-O-mPEG ₅ -nalbuphine (5) (n = 5) (55 mg). Got.

(Synthesis of _{6-O-mPEG 6 -3-} O-MEM- nalbuphine (4) (n = 6) )

3-O-MEM-Nalbuphine (3) (77.6 mg, 0.17 mmol) and mPEG ₆ -OMs (199 mg, 0.53 mmol) were dissolved in toluene (20 mL). The mixture was concentrated to remove about 12 mL of toluene. Anhydrous DMF (0.2 mL) was added followed by NaH (60% dispersion in mineral oil, 41 mg, 1.03 mmol). The resulting mixture was heated at 45 ° C. for 23 hours before additional NaH (46 mg) was added. The mixture was heated at 45 ° C. for an additional 24 hours. When the mixture was cooled to room temperature, saturated aqueous NaCl (5 mL) was added to quench the reaction. The mixture was diluted with water (10 mL) and extracted with EtOAc (4 × 15 mL). The combined organic solution was washed with brine, dried over Na ₂ SO ₄ and concentrated. The residue was used directly in the next step.

(Synthesis of 6-O-mPEG ₆ -Nalbuphine (5) (n = 6))

The above _{6-O-mPEG 6 -3-} O-MEM- nalbuphine (4), 20 hours at room temperature, was stirred in 2M HCl in methanol (30 mL). The mixture was diluted with water (5 mL) and concentrated to remove methanol. The aqueous solution was adjusted to pH 9.30 with 1N NaOH and extracted with dichloromethane (5 × 20 mL). The combined organic solution was washed with brine, dried over Na ₂ SO ₄ and concentrated. The residue was dissolved in DCM (about 1 mL). 1N HCl in ether (20 mL) was added and centrifuged. The residue was collected and redissolved in DCM (40 mL). The DCM solution was washed with 5% aqueous NaHCO ₃ (2 × 20 mL), water (30 mL), brine (2 × 30 mL), dried over Na ₂ SO ₄ , concentrated, and the product 6-O-mPEG ₆ -Nalbuphine (5) (n = 6) (68 mg) was obtained.

_{(6-O-mPEG 7 -3} -O-MEM- nalbuphine Synthesis of (4, n = 7))

A 50 mL round bottom flask was charged with 3-O-MEM-nalbuphine (3) (82.8 mg, 0.186 mmol), mPEG ₇ -Br (151 mg, 0.46 mmol), and toluene (15 mL). The mixture was concentrated to remove about 9 mL of toluene. Anhydrous DMF (0.2 mL) was added. NaH (60% dispersion in mineral oil, 50 mg, 1.25 mmol) was added. The resulting mixture was heated at 45 ° C. for 22.5 hours before additional NaH (38 mg, 0.94 mmol) was added. The mixture was heated at 45 ° C. for an additional 5 hours. When the mixture was cooled to room temperature, saturated aqueous NaCl (5 mL) was added to quench the reaction. The mixture was diluted with water (10 mL) and extracted with EtOAc (4 × 10 mL). The combined organic solution was washed with brine, dried over Na ₂ SO ₄ and concentrated. The residue was used directly in the next step.

(Synthesis of 6-O-mPEG ₇ -Nalbuphine (5) (n = 7))

The above _{6-O-mPEG 7 -3-} O-MEM- nalbuphine (4), 20 hours at room temperature, was stirred in 2M HCl in methanol (20 mL). The mixture was diluted with water and concentrated to remove methanol. The aqueous solution was adjusted to pH 9.30 with NaHCO ₃ and 0.2N NaOH and extracted with dichloromethane (4 × 20 mL). The combined organic solution was washed with brine, dried over Na ₂ SO ₄ and concentrated. The residue was purified by flash column chromatography on silica gel, washed with DCM under acidic conditions, adjusted to pH 9.35 and extracted with DCM. The product was still contaminated with low molecular weight PEG. The residue was dissolved in DCM (about 2 mL). 1N HCl in ether (10 mL) was added and centrifuged. The residue was collected and redissolved in DCM (10 mL). The DCM solution was washed with 5% aqueous NaHCO ₃ solution, brine, dried over Na ₂ SO ₄ and concentrated to give the product 6-O-mPEG ₇ -nalbuphine (5) (n = 7) (49 mg). Obtained.

(Synthesis of _{6-O-mPEG 8 -3-} O-MEM- nalbuphine (4) (n = 8) )

A 50 mL round bottom flask was charged with 3-O-MEM-nalbuphine (3) (80.5 mg, 0.181 mmol), mPEG ₈ -Br (250 mg, 0.56 mmol), and toluene (15 mL). The mixture was concentrated to remove about 6 mL of toluene. Anhydrous DMF (0.2 mL) was added. NaH (60% dispersion in mineral oil, 49 mg, 1.23 mmol) was added. The resulting mixture was heated at 45 ° C. for 23 hours, the mixture was cooled to room temperature, saturated aqueous NaCl (5 mL) and water (10 mL) were added to quench the reaction. The mixture was extracted with EtOAc (4 × 20 mL). The combined organic solution was washed with brine, dried over Na ₂ SO ₄ and concentrated. The residue was used directly in the next step.

(Synthesis of 6-O-mPEG ₈ -Nalbuphine (5) (n = 8))

The above _{6-O-mPEG 8 -3-} O-MEM- nalbuphine (4), 17 hours at room temperature, was stirred in 2M HCl in methanol (20 mL). The mixture was diluted with water and concentrated to remove methanol. The aqueous solution was adjusted to pH 9.32 with NaHCO ₃ and 0.2N NaOH and extracted with dichloromethane (4 × 20 mL). The combined organic solution was washed with brine, dried over Na ₂ SO ₄ and concentrated. The residue was dissolved in DCM (about 1 mL). 1N HCl in ether (20 mL) was added and centrifuged. The residue was collected and redissolved in DCM (30 mL). The DCM solution was washed with 5% aqueous NaHCO ₃ (60 mL), water (30 mL), brine (30 mL), dried over Na ₂ SO ₄ and concentrated. The residue was purified by flash column chromatography on silica gel using 0-10% MeOH in dichloromethane to give the product 6-O-mPEG ₈ -nalbuphine (5) (n = 8) (78.4 mg )

(Synthesis of _{6-O-mPEG 9 -3-} O-MEM- nalbuphine (4) (n = 9) )

A 50 mL round bottom flask was charged with 3-O-MEM-nalbuphine (3) (120 mg, 0.27 mmol), mPEG ₉ -OMs (245 mg, 0.48 mmol), and toluene (20 mL). The mixture was concentrated to remove about 10 mL of toluene. NaH (60% dispersion in mineral oil, 63 mg, 1.57 mmol) was added followed by anhydrous DMF (0.5 mL). The resulting mixture was heated at 45 ° C. for 17 hours. More NaH (60% dispersion in mineral oil, 60 mg) was added based on the HPLC results, after which the mixture was heated at 45 ° C. for an additional 5.5 hours. The mixture was cooled to room temperature and saturated aqueous NaCl (2 mL) and water (15 mL) were added to quench the reaction. The mixture was extracted with EtOAc (4 × 20 mL). The combined organic solution was washed with brine, dried over Na ₂ SO ₄ and concentrated. The residue was purified by flash column chromatography on silica gel using 3-8% methanol in biotage to give the product 6-O-mPEG ₉ -nalbuphine (207 mg) in 90% yield. Got.

(Synthesis of 6-O-mPEG ₉ -Nalbuphine (5) (n = 9))

Above _{6-O-mPEG 9 -3-} O-MEM- nalbuphine (4) (207mg, 0.24mmol) and 17 h at room temperature, was stirred in 2M HCl in methanol (33 mL). The mixture was diluted with water and concentrated to remove methanol. The aqueous solution was adjusted to pH 9.16 with 1N NaOH and extracted with dichloromethane (4 × 25 mL). The combined organic solution was washed with brine, dried over Na ₂ SO ₄ and concentrated. The residue was purified by flash column chromatography on silica gel using 3-8% methanol in dichloromethane to give the product 6-O-mPEG ₉ -nalbuphine (4) (n = 9) (129.3 mg) was obtained.

(Example 3)
(Preparation of oligomer-nalbuphine complex- "Method C")
PEG-nalbuphine was prepared using a third approach. Schematically, the technique applied to the present embodiment is shown below.

Synthesis of TrO-PEG ₅ -OH (7) (n = 5)

PEG ₅ -di-OH (6) (n = 5) (5.88 g, 24.19 mmol) was dissolved in toluene (30 mL) and concentrated under reduced pressure to remove toluene. The residue was dried under high vacuum. Anhydrous DMF (40 mL) was added followed by DMAP (0.91 g, 7.29 mmol) and TrCl (trityl chloride) (1.66 g, 5.84 mmol). The resulting mixture was heated at 50 ° C. for 22 hours. The mixture was concentrated to remove the solvent (high vacuum, 50 ° C.). The residue was mixed with water and extracted with EtOAc (3 × 25 mL). The combined organic solution was washed with brine, dried over Na ₂ CO ₃ and concentrated. The residue was purified by flash column chromatography on silica gel to give 1.29 g of product in 46% yield. The product was confirmed by ¹ H-NMR in CDCl ₃ .

(Synthesis of TrO-PEG _n -OH (7) (n = various values))

Following the procedure similar to the preparation of _TrO-PEG 5 -OH, the other _TrO-PEG n -OH, the corresponding PEG _n - was synthesized from di -OH.

(Synthesis of TrO-PEG ₅ -OMs (8) (n = 5))

Methanesulfonyl chloride (0.35 mL, 4.48 mmol) was added to TrO-PEG ₅ -OH (8) (n = 5) (1.29 g, 2.68 mmol) in dichloromethane (15 mL) and triethylamine (0.9 mL, 6.46 mmol) was added dropwise at 0 ° C. After the addition, the resulting solution was stirred at room temperature for 16.5 hours. Water was added to stop the reaction. The organic phase was separated and the aqueous solution was extracted with dichloromethane (10 mL). The combined organic solution was washed with brine (3 × 30 mL), dried over Na ₂ SO ₄ and concentrated to give the product (1.16 g) as an oil in 78% yield. The product (8) (n = 5) was confirmed by ¹ H-NMR in CDCl ₃ .

(Synthesis of TrO-PEG _n -OMs (8) (n = various values))

Following similar procedures for the preparation of TrO-PEG ₅ -OMs, other TrO-PEG _n -OMs were synthesized from the corresponding TrO-PEG _n -OH.

(Synthesis of 3-O-MEM-6-O-TrO-PEG ₄ -nalbuphine (9) (n = 4))

In a round bottom flask, 3-O-MEM-nalbuphine (3) (120 mg, 0.27 mmol) [prepared according to the synthesis of compound (3) provided in Example 2], TrO-PEG ₄ -OMs (8 ) (N = 4) (143.4 mg, 0.28 mmol), and toluene (40 mL). The mixture was concentrated to remove about 30 mL of toluene. NaH (60% dispersion in mineral oil, 150 mg, 3.75 mmol) was added followed by anhydrous DMF (0.2 mL). The resulting mixture was heated at 45 ° C. for 4.5 hours. More NaH (60% dispersion in mineral oil, 146 mg) was added and the mixture was stirred at 45 ° C. for an additional 18 hours. The mixture was cooled to room temperature, saturated with aqueous NaCl (2 mL), and water (15 mL) was added to quench the reaction. The mixture was extracted with EtOAc (4 × 20 mL). The combined organic solution was washed with brine, dried over Na ₂ SO ₄ and concentrated. The residue was purified by flash column chromatography on silica gel using 0-10% methanol in dichloromethane (Biotage) to give the product 3-O-MEM-6-O-TrO-PEG ₄ -nalbuphine ( 9) (n = 4) (about 150 mg) was obtained.

(Synthesis of 6-O-HO-PEG ₄ -nalbuphine (10) (n = 4))

Above _{6-O-TrO-PEG 4} -3-O-MEM- nalbuphine a (9) (n = 4) (150mg), was stirred in 2M HCl in 1 day methanol at room temperature (12 mL). The mixture was diluted with water and concentrated to remove methanol. The aqueous solution was adjusted to pH 9.08 with NaOH and extracted with EtOAc (3 × 20 mL). The combined organic solution was washed with brine, dried over Na ₂ SO ₄ and concentrated. The residue was purified by flash column chromatography on silica gel to give the product 6-O—OH-PEG ₄ -nalbuphine (10) (n = 4) (26.9 mg). The product was confirmed by ¹ H-NMR, LC-Ms, HPLC.

(Synthesis of 3-O-MEM-6-O-TrO-PEG ₅ -nalbuphine (9) (n = 5))

In a round bottom flask, 3-O-MEM-nalbuphine (3) (318 mg, 0.71 mmol) [prepared according to the synthesis of compound (3) provided in Example 2], TrO-PEG ₅ -OMs (8 ) (N = 5) (518.5 mg, 0.93 mmol), and toluene (100 mL). The mixture was concentrated to remove about 75 mL of toluene. NaH (60% dispersion in mineral oil, 313 mg, 7.8 mmol) was added followed by anhydrous DMF (1.0 mL). The resulting mixture was stirred at room temperature for 30 hours and then at 60 ° C. for 19.5 hours. The mixture was cooled to room temperature, saturated with aqueous NaCl (5 mL), and water (5 mL) was added to quench the reaction. The organic phase was separated and the aqueous solution was extracted with EtOAc. The combined organic solution was washed with brine, dried over Na ₂ SO ₄ and concentrated. The residue was purified by flash column chromatography on silica gel using 0-10% methanol in dichloromethane (Biotage) to give the product 3-O-MEM-6-O-TrO-PEG ₅ -nalbuphine ( 718 mg) was obtained. The product (9) (n = 5) was impure and was used in the next step without further purification.

(Synthesis of 6-O-HO-PEG ₅ -nalbuphine (10) (n = 5))

Above _{6-O-TrO-PEG 5} -3-O-MEM- nalbuphine a (9) (n = 5) (718mg), 19 hours at room temperature, was stirred in 2M HCl in methanol (30 mL). The mixture was diluted with water and concentrated to remove methanol. The aqueous solution was adjusted to pH 9.16 with NaOH and extracted with DCM (3 × 20 mL). The combined organic solution was washed with brine, dried over Na ₂ SO ₄ and concentrated. The residue was purified twice by flash column chromatography on silica gel to give a very pure product 6-O-OH-PEG ₅ -nalbuphine 10 (n = 5) (139 mg) less pure product (48 mg ) The product was analyzed by ¹ H-NMR, LC-Ms, HPLC.

Example 4
(Receptor binding of PEG-nalbuphine conjugate)
Using conventional receptor binding assay techniques, several molecules were analyzed to determine the binding activity of the opioid receptor at the kappa, mu, and delta opioid subtypes.

Briefly, the receptor binding affinity of nalbuphine and PEG-nalbuphine conjugates was measured using a radioligand binding assay in CHO cells that heterologously express recombinant human mu, delta, or kappa opioid receptors. Cells were seeded in 24-well plates at a density of 0.2-0.3 × 10 ⁶ cells / well and washed with assay buffer (pH 7.4) containing 50 mM Tris HCl and 5 mM MgCl ₂ . Competitive binding assays were performed on whole cells incubated with increasing concentrations of test compound in the presence of the appropriate concentration of radioligand. 0.5 nM ³ H naloxone, 0.5 nM ³ H diprenorphine, and 0.5 nM ³ H DPDPE were used as competing radioligands for the mu, kappa, and delta receptors, respectively. Incubations were performed for 2 hours at room temperature using triplicate wells at each concentration. At the end of incubation, cells were washed with 50 mM Tris HCl (pH 8.0), lysed with NaOH, and bound radioactivity was measured using a scintillation counter.

Specific binding was determined by reducing cpm bound in the presence of a 50-100 fold excess of non-radioactive ligand. The binding data assay is analyzed using GraphPad Prism 4.0 and IC ₅₀ is generated by non-linear regression from a dose-response curve. Ki values were calculated as follows using the Cheng Prusoff equation with Kd values from saturation isotherms: Ki = IC ₅₀ / (1+ [ligand] / Kd).

The PEG conjugate of nalbuphine retains binding affinity for the opioid receptor. Table 1 shows the binding affinity (Ki (nM)) of PEG conjugates of nalbuphine at mu, delta, and kappa opioid receptors. The loss of binding affinity after PEG conjugation is less than 15 times the loss of binding affinity of the parent nalbuphine in all three receptor subtypes. See Table 1. PEG conjugation results in a 10-15 fold loss in binding affinity at mu and kappa opioid receptors, but not at delta opioid receptors. The binding affinity at the delta opioid receptor is comparable to or even greater than the binding affinity of the parent nalbuphine. Please refer to FIG. The differential change in binding affinity of the three opioid receptor subtypes suggests that the receptor selectivity of the nalbuphine conjugate is altered compared to the parent nalbuphine.

(Example 5)
(Preparation of U50488 conjugate)
PEG-U50488 can be prepared according to the procedure outlined below.

Example _5a: Synthesis of mPEG n -O-U50488 conjugate

The mPEGn-O-U504488 conjugate can be prepared according to the general schematic provided below.

((+/-)-trans-2- (pyrrolidin-1-yl) cyclohexanol ((+/-)-1))
Pyrrolidine (4.26 g, 60 mmol) was added to a solution of cyclohexene oxide (1.96 g, 20 mmol) in H ₂ O (6 mL) and the resulting mixture was heated at 90 ° C. for 16 hours. After cooling, the solvent was removed under reduced pressure and the resulting residue was extracted with DCM (10 mL × 3). The organic phases were combined and dried using anhydrous Na ₂ SO ₄ . After removal of Na ₂ SO ₄ by filtration, the solvent was evaporated and the material was dried under vacuum to give the desired compound (+/−)-1 (3.3 g, 19.5 mmol, 98% yield). Obtained. ¹ H NMR (CDCl ₃ ) δ 4.06 (s, 1H), 3.45-3.30 (m, 1H), 2.75-2.63 (m, 2H), 2.62-2.50 ( m, 2H), 2.51-2.42 (m, 1H), 1.90-1.58 (m, 8H), 1.45-1.15 (m, 4H).

((+/-)-trans-N-methyl-2- (pyrrolidin-1-yl) cyclohexaneamine ((+/-)-2))
To a solution of compound (+/−)-1 (1.01 g, 6 mmol) and TEA (0.98 mL, 7 mmol) in DCM (20 mL) was added methanesulfonyl chloride (0.51 mL, 6.6 mmol) dropwise. Added. The reaction mixture was stirred at room temperature for 2 hours. The solvent was removed under reduced pressure and 5 mL of methylamine (40% aqueous solution) was added to room temperature. The solution was stirred at room temperature for a further 16 hours. The reaction mixture was then added to DCM (50 mL) and washed with saturated NaHCO ₃ solution (2 × 25 mL). The organic phases were combined and dried over anhydrous Na ₂ SO ₄ . After removal of Na ₂ SO ₄ by filtration, the solvent was removed and the material was dried under vacuum to give the desired compound (+/−)-2 (1.0 g, 5.5 mmol, 91% yield). It was. ¹ H NMR (CDCl ₃ ) δ 2.61 (s, 1H), 2.49-2.30 (m, 6H), 2.29 (s, 3H), 2.09-1.96 (m, 2H) 1.67-1.52 (m, 7H), 1.11-1.02 (m, 3H).

((+/-)-N-methyl-N- [2- (pyrrolidin-1-yl) cyclohexyl] -2- (3-chloro-4-hydroxyphenyl) acetamide ((+/-)-4))
3-Chloro-4-hydroxyphenylacetic acid (186 mg, 1 mmol) and NHS (115 mg, 3 mmol) were dissolved in DCM (20 mL). Then DCC (1.1 mmol) was added to the solution and the reaction mixture was stirred at room temperature for 16 hours during which time a precipitate formed. After removing the precipitate by filtration, the resulting filtrate was mixed with compound (+/−) 3 (182 mg, 1 mmol). The resulting solution was stirred overnight at room temperature. The solvent was removed under reduced pressure and the resulting residue was subjected to flash chromatography purification (MeOH / DCM = 2% -18%) to give the desired product (+/−)-4 (120 mg, 0.34 mmol, Yield 34%). ¹ H NMR (CDCl ₃ ) δ 7.12 (s, 1H), 7.00-6.86 (m, 2H), 4.56 (s, 1H), 3.66-3.61 (m, 2H) 3.09-2.85 (m, 6H), 2.85 (s, 3H), 1.71-1.45 (m, 8H), 1.44-1.11 (m 4H). LC / MS 351.1 [M + H] ^< +>.

((+/-)-N-methyl-N- [2- (pyrrolidin-1-yl) cyclohexyl] -2- (3-chloro-4-methoxy-tri (ethylene glycol) phenyl) acetamide ((+/- ) -5a))
Compound (+/−)-4 (50 mg, 0.14 mmol), methoxytri (ethylene glycol) methanesulfonate (48.4 mg, 0.2 mmol), and anhydrous K ₂ CO ₃ (70 mg, 0.5 mmol) in acetone ( 10 mL). The resulting mixture was stirred at reflux for 16 hours. The resulting residue was subjected to flash chromatography purification (MeOH / DCM = 2% -15%) to give the desired compound (+/−)-5a (30 mg, 0.06 mmol, 43% yield). ¹ H NMR (CDCl ₃ ) δ 7.31 (s, 1H), 7.18 (d, 1H), 7.06 (d, 1H), 4.61-4.51 (m 1H), 4.19 ( t, 2H), 3.90 (t, 2H), 3.76-3.44 (m, 10H), 3.36 (s, 3H), 2.95-2.83 (m, 6H), 2 11-1.19 (m, 12H). LC / MS 497.2 [M + H] ⁺

((+/-)-N-methyl-N- [2- (pyrrolidin-1-yl) cyclohexyl] -2- (3-chloro-4-methoxy-penta (ethylene glycol) phenyl) acetamide) ((+ / -)-5b)
Compound (+/−)-4 (80 mg, 0.23 mmol), methoxypenta (ethylene glycol) methanesulfonate (108.9 mg, 0.33 mmol), and anhydrous K ₂ CO ₃ (112 mg, 0.8 mmol) in acetone (15 mL). The mixture was stirred at reflux for 16 hours. The solid was removed by filtration and the solvent was evaporated under reduced pressure. The resulting residue was subjected to flash chromatography purification (MeOH / DCM = 2% to 15%) to give the desired compound (+/−)-5a (60 mg, 0.10 mmol, 45% yield). ¹ H NMR (CDCl ₃ ) δ 7.25 (s, 1H), 7.10 (d, 1H), 6.85 (d, 1H), 4.55-4.45 (m 1H), 4.14 ( t, 2H), 3.87 (t, 2H), 3.81-3.42 (m, 18H), 3.37 (s, 3H), 2.90-2.35 (m, 6H), 2 0.09-1.15 (m, 12H). LC / MS 585.3 [M + H] ^< +>.

((+/-)-N-methyl-N- [2- (pyrrolidin-1-yl) cyclohexyl] -2- (3-chloro-4-methoxy-hexa (ethylene glycol) phenyl) acetamide ((+/- ) -5c))
Compound (+/−)-4 (50 mg, 0.23 mmol), methoxyhexa (ethylene glycol) methanesulfonate (150 mg, 0.37 mmol), and anhydrous K ₂ CO ₃ (112 mg, 0.8 mmol) in acetone (15 mL ). The mixture was stirred at reflux for 16 hours. The solid was removed by filtration and the solvent was evaporated under reduced pressure. The resulting residue was subjected to flash chromatography purification (MeOH / DCM = 2% -15%) to give the desired compound (+/−)-5c (61 mg, 0.09 mmol, 39% yield). ¹ H NMR (CDCl ₃ ) δ 7.27 (s, 1H), 7.16 (d, 1H), 6.88 (d, 1H), 4.72-4.51 (m 1H), 4.16 ( t, 2H), 3.89 (t, 2H), 3.79-3.49 (m, 26H), 3.38 (s, 3H), 3.18-2.53 (m, 6H), 2 .10-1.12 (m, 12H). LC / MS 673.4 [M + H] ^< +>.

  Example 5B: Synthesis of di-mPEGn-CH2-U50488 conjugate

Di-mPEGn-CH2-U504488 conjugates can be prepared according to the general schematic provided below.

(3-Cyclohexene 1,1-dimethanol methoxytri (ethylene glycol) ether (2a))
3-Cyclohexene 1,1-dimethanol 1 (284 mg, 2 mmol) was dissolved in anhydrous DMF (6 mL). At room temperature, NaH (60% in mineral oil, 320 mg, 8 mmol) was added and the solution was stirred for an additional 10 minutes. Methoxytri (ethylene glycol) mesylate (1.21 g, 5 mmol) was added to the solution. After the reaction solution was stirred at 45 ° C. for 18 h, saturated NH ₄ Cl solution (30 mL) was added to the solution. The solution was extracted with EtOAc (20 mL × 2). The organic phases were combined, dried over Na ₂ SO ₄ , filtered, and the solvent was removed under reduced pressure to give compound 2a (850 mg, 1.96 mmol, 98% yield). ¹ H NMR (CDCl ₃ ) δ 5.62-5.58 (m, 2H), 3.66-3.51 (m, 24H), 3.38 (s, 6H), 3.34 (d, 2H) , 3.25 (d, 2H), 2.01 (m, 2H), 1.87 (m, 2H), 1.52 (t, 2H). LC / MS 435 [M + H] ⁺ , 452 [M + NH ₄ ] ⁺ , 457 [M + Na] ⁺ .

(3-Cyclohexene 1,1-dimethanol methoxydi (ethylene glycol) ether (2b))
3-Cyclohexene 1,1-dimethanol (426 mg, 3 mmol) 1 was dissolved in anhydrous DMF (9 mL). At room temperature, NaH (60% in mineral oil, 480 mg, 12 mmol) was added and the solution was stirred for an additional 10 minutes. Methoxydi (ethylene glycol) mesylate (1.5 g, 7.5 mmol) was added to the solution. After the reaction solution was stirred at 45 ° C. for 18 h, saturated NH ₄ Cl solution (30 mL) was added to the solution. The solution was extracted with EtOAc (20 mL × 2). The organic phases were combined, dried over Na ₂ SO ₄ , filtered, and the solvent was removed under reduced pressure to give compound 2b (1.18 g, 2.9 mmol, 98% yield). ¹ H NMR (CDCl ₃ ) δ 5.64-5.56 (m, 2H), 3.66-3.54 (m, 16H), 3.35 (s, 6H), 3.33 (d, 2H) 3.28 (d, 2H), 1.99 (m, 2H), 1.87 (m, 2H), 1.53 (t, 2H).

(3,3-di [methoxytri (ethylene glycol) methyl] -7-oxabicyclo [4.1.0] heptane (3a))
Compound 2a (850 mg, 1.96 mmol) was dissolved in DCM (20 mL). At room temperature, mCPBA (up to 77%, 0.75 g, about 3 mmol) was added to the solution. The reaction mixture was stirred at room temperature for 3.5 hours. Saturated Na ₂ S ₂ O ₃ solution (10 mL) was added to the solution resulting in an additional 10 minutes of stirring. The resulting solution was extracted with DCM (20 mL × 2). The organic phases were combined, dried over Na ₂ SO ₄ , filtered, and the solvent was removed under reduced pressure to give compound 3a (870 mg, 1.86 mmol, 95% yield). ¹ H NMR (CDCl ₃ ) δ 3.66-3.55 (m, 24H), 3.38 (s, 6H), 3.27-3.12 (m, 6H), 1.99-1.67 ( m, 6H). LC / MS 451 [M + H] ⁺ , 468 [M + NH ₄ ] ⁺ , 473 [M + Na] ⁺ .

(3,3-di [methoxydi (ethylene glycol) methyl] -7-oxabicyclo [4.1.0] heptane (3b))
Compound 2b (1.18 g, 3.41 mmol) was dissolved in DCM (20 mL). At room temperature, mCPBA (up to 77%, 1.3 g, ca. 5.2 mmol) was added to the solution. The reaction solution was stirred at room temperature for 3.5 hours. Saturated Na ₂ S ₂ O ₃ solution (15 mL) was added to the solution resulting in an additional 10 minutes of stirring. The resulting solution was extracted with DCM (20 mL × 2). The organic phases were combined, dried over Na ₂ SO ₄ , filtered and the solvent was removed under reduced pressure to give compound 3b (1.27 g, 3.12 mmol, 92% yield). ¹ H NMR (CDCl ₃ ) δ 3.65-3.54 (m, 24H), 3.38 (s, 6H), 3.27-3.10 (m, 6H), 2.00-1.68 ( m, 6H).

(Trans (+/−)-4 or 5-di [methoxytri (ethylene glycol) methyl] -2- (1-pyrrolidinyl) -cyclohexanol (4a))
Compound 3a (870 mg, 1.93 mmol) and pyrrolidine (2.5 mL) were added to water (8 mL) and heated at reflux for 5 hours. The resulting solution was extracted with DCM (10 mL × 2). The organic phases were combined, dried over Na ₂ SO ₄ , filtered and the solvent removed under reduced pressure to give 4a as a mixture of 4ax and 4ay (total 910 mg). The product was used in the next reaction without further purification.

(Trans-(+/-)-4 or 5-di [methoxydi (ethylene glycol) methyl] -2- (1-pyrrolidinyl) -cyclohexanol (4b))
Compound 3b (1.27 g, 3.12 mmol) and pyrrolidine (2.5 mL) were added to water (8 mL) and heated at reflux for 5 hours. The resulting solution was extracted with DCM (10 mL × 2). The organic phases were combined, dried over Na ₂ SO ₄ , filtered and the solvent removed under reduced pressure to give 4b as a mixture of 4bx and 4by (total 1.3 g). The product was used in the next reaction without further purification.

(Trans (+/-)-4- or 5-di [methoxytri (ethylene glycol) methyl] -N-methyl-2- (1-pyrrolidinyl) -cyclohexaneamine (5a))
Compound 4a (910 mg, 1.74 mmol) was dissolved in DCM (20 mL) and TEA (0.42 mL, 3 mmol) was added. At room temperature, methanesulfonyl chloride (0.16 mL, 2 mmol) was added dropwise. After stirring was continued overnight at room temperature, the resulting mixture was evaporated under reduced pressure. The resulting residue was dissolved in methylamine (40% (w / w) in water, 6 mL) and the solution was stirred at room temperature for 30 hours. The solution was then extracted with DCM (10 mL × 2). The organic phases were combined, dried over Na ₂ SO ₄ , filtered and the solvent removed under reduced pressure to give 5a as a mixture of 5ax and 5ay. The product was used in the next reaction without further purification.

(Trans-(+/-)-4 or -5-di [methoxydi (ethylene glycol) methyl] -N-methyl-2- (1-pyrrolidinyl) -cyclohexaneamine (5b))
Compound 4b (1.3 g, 3 mmol) was dissolved in DCM (20 mL) and TEA (0.84 mL, 6 mmol) was added. At room temperature, methanesulfonyl chloride (0.28 mL, 3.5 mmol) was added dropwise. After stirring was continued overnight at room temperature, the resulting mixture was evaporated under reduced pressure. The resulting residue was dissolved in methylamine (40% (w / w) in water, 6 mL) and the solution was stirred at room temperature for 30 hours. The resulting solution was then extracted with DCM (10 mL × 2). The organic phases were combined, dried over Na ₂ SO ₄ , filtered and the solvent removed under reduced pressure to give 5b as a mixture of 5bx and 5by. The product was used in the next reaction without further purification.

(Trans-(+/-)-4 or -5-di [methoxytri (ethylene glycol) methyl] -N-methyl-N- [2- (pyrrolidin-1-yl) cyclohexyl] -2- (3,4 (Dichloro) acetamide (6a))
3,4-Dichlorophenylacetic acid (410 mg, 2 mmol), compound 5a (926 mg, 1.74 mmol), and DMAP (10 mg) were dissolved in DCM (10 mL). Then DCC (515 mg, 2.5 mmol) was added to the solution and the reaction mixture was stirred at room temperature for 4 hours, during which time a precipitate formed. After removing the precipitate by filtration, the resulting filtrate solvent is evaporated and the residue is subjected to flash chromatography purification (MeOH / DCM = 2% -8%) to give the desired product 6a in a mixture of 6ax and 6ay. (Total 477 mg, 0.34 mmol, yield 20%). ¹ H NMR (CDCl 3) δ 7.39-7.33 (m, 2H), 7.15-7.10 (m, 1H), 3.71-3.51 (m, 28H), 3.42-3 .16 (m, 3H), 3.35 (s, 6H), 2.82, 2.79 (total s, s, 3H, ratio 3: 5), 2.70-2.30 (m, 5H) , 1.73-1.17 (m, 11H). LC / MS 721 [M + H] ⁺ , 743 [M + Na] ⁺ .

(Trans (+/-)-4 or -5-di [methoxydi (ethylene glycol) methyl] -N-methyl-N- [2- (pyrrolidin-1-yl) cyclohexyl] -2- (3,4-dichloro Acetamide (6b))
3,4-Dichlorophenylacetic acid (707 mg, 3.45 mmol), compound 5b (1.33 g, 2.98 mmol), and DMAP (10 mg) were dissolved in DCM (10 mL). Next, DCC (865 mg, 4.2 mmol) was added to the solution and the reaction mixture was stirred at room temperature for 4 hours, during which time a precipitate formed. After removing the precipitate by filtration, the resulting filtrate solvent is evaporated and the residue is subjected to flash chromatography purification (MeOH / DCM = 2% -8%) to give the desired product 6b in a mixture of 6bx and 6by. (Total 306 mg, 0.49 mmol, 16% yield). ¹ H NMR (CDCl 3) δ 7.33-7.27 (m, 2H), 7.06-7.04 (m, 1H), 3.65-3.46 (m, 20H), 3.42-3 .12 (m, 3H), 3.37 (s, 6H), 2.76, 2.74 (total s, s, 3H, ratio 1.1: 1), 2.72-2.24 (m, 5H), 1.71-1.07 (m, 11H). LC / MS 633 [M + H] ⁺ , 655 [M + Na] ⁺ .

(Example 6)
(Preparation of oligomer-U69593 conjugate)
PEG-U69593 can be prepared according to the procedure outlined below. Conventional organic synthesis techniques are used in performing the approach.

(Example 7)
(Preparation of conjugate using other than nalbuphine, U50488, and U69593)
Conjugates of opioid agonists other than nalbuphine, U50488, and U69593 can be prepared, wherein the opioid agonist of formula I is replaced by nalbuphine, U50488, and U69593 as described in Example 1 General synthetic schemes and procedures can be followed.

(Example 8)
(In vitro efficacy of PEG-nalbuphine conjugates)
The in vitro efficacy of PEG-nalbuphine conjugates was determined using a GTPγS binding assay in CHO cells expressing recombinant human mu or delta opioid receptors, or HEK cells expressing recombinant human kappa opioid receptors. . Test compounds and / or vehicle were preincubated with cell membranes and 3 μM GDP in modified HEPES buffer (pH 7.4) for 20 minutes, followed by addition of SPA beads at 30 ° C. for an additional 60 minutes. The reaction is initiated with 0.3 nM [ ³⁵ S] GTPγS for an additional 30 minutes incubation time. A test compound-induced increase in [ ³⁵ S] GTPγS binding of more than 50 percent (≧ 50%) to the receptor subtype-specific jaw response indicates possible opiate receptor agonist activity. 0.1 μM DPDPE, 1 μM U-69593, and 1 μM DAMGO were used as agonists specific for the delta opioid receptor, kappa opioid receptor, and mu opioid receptor, respectively. Opioid receptor antagonist activity was measured using an inhibition of agonist-induced increase of more than 50 percent (≧ 50%) of the [ ³⁵ S] GTPγS binding response. Nalbuphine, 6-O-mPEG ₃ -Nalbuphine, 6-O-mPEG ₆ -Nalbuphine, 6-O-mPEG ₉ -Nalbuphine in both agonist and antagonist modes 10, 1, 0.1, 0.01, And screened at a concentration of 0.001 μM. EC ₅₀ values or IC ₅₀ values were calculated from dose-response curves as a measure of the agonist or antagonist activity of the test compound, respectively.

Table 2 shows EC ₅₀ / IC ₅₀ values for nalbuphine and PEG-nalbuphine conjugates that activate or inhibit GTPγS binding and thus reflect their agonist or antagonist activity. PEG-nalbuphine conjugates are full agonists of kappa opioid receptors and antagonists of mu opioid receptors, similar to the pharmacological properties of nalbuphine. _{6-O-mPEG 3 - EC} 50 of nalbuphine are similar to _{EC 50} for nalbuphine kappa opioid receptor, suggesting that there is no loss of effectiveness in the PEG size. Beyond a PEG size of 3, the effectiveness of PEG-nalbuphine conjugates at the kappa opioid receptor is shown by increasing EC ₅₀ values of 6-O-mPEG ₆ -nalbuphine and 6-O-mPEG ₉ -nalbuphine. As a function of PEG size. PEG-Nalbuphine appeared to have a mu opioid receptor antagonist potency comparable to that of the parent nalbuphine. At the delta opioid receptor, 6-O-mPEG ₉ -nalbuphine acted as a weak antagonist, whereas nalbuphine, 6-O-mPEG ₃ -nalbuphine, 6-O-mPEG ₆ -nalbuphine did not interact with the delta opioid receptor. Had no effect.

Example 9
(Permeability in vitro)
The in vitro permeability of PEG-nalbuphine conjugates was measured using a bidirectional transport assay in Caco-2 cells. PEG-nalbuphine conjugate was added at a concentration of 10 μM to either the apical or basolateral compartment of the Caco-2 monolayer and incubated for 2 hours. At the end of the incubation, the concentration of the apical and basolateral compartments was measured using LC-MS. Permeability was calculated as P _app = J / A × C ₀ , where P _app is the apparent permeability (cm / s), J is the flow (mole / second), and A is The surface area (cm ² ) of the insert, and C ₀ is the initial concentration (mol / cm ³ ).

FIG. 2a shows the in vitro apparent permeability of PEG-nalbuphine conjugates measured in Caco-2 cells in the AB (apical-basal) and B-A (baso-apical) directions. Show. The permeability at AB and, to a lesser extent, at BA, decreases with increasing PEG chain length. AB permeability representing mucosal-serosa transport in vivo is greater than 1 × 10 ⁻⁶ cm / sec for all compounds and nalbuphine and PEG-nalbuphine conjugates will be well absorbed orally It shows that.

FIG. 2b shows the PEG-nalbuphine conjugate efflux ratio calculated as the ratio of BA / AB permeability. Larger efflux ratios than single bodies reflect transport asymmetry in the apical-basolateral direction, suggesting a role for transporters in overall permeability. The effluent ratio for the parent nalbuphine is close to 1, indicating that it is not a promising substrate for the transporter. However, 6-O-mPEG ₅ -nalbuphine, 6-O-mPEG ₆ -nalbuphine, 6-O-mPEG ₇ -nalbuphine, 6-O-mPEG ₈ -nalbuphine, and 6-O-mPEG ₉ -nalbuphine are 3 Has a greater efflux ratio and is therefore a promising substrate for efflux transporters in Caco-2 cells.

(Example 10)
(In vivo brain penetration of PEG-nalbuphine conjugate)
The ability of PEG-nalbuphine conjugates to cross the blood brain barrier (BBB) and enter the central nervous system was measured using the brain: plasma ratio in rats. Briefly, rats were injected intravenously with 25 mg / kg nalbuphine, PEG-nalbuphine conjugate, or atenolol. One hour after injection, the animals were sacrificed and plasma and brain were collected and immediately frozen. Following tissue and plasma removal, the concentration of the compound in the brain and plasma was measured using LC-MS / MS. The brain: plasma ratio was calculated as the ratio of concentrations measured in the brain and plasma. Atenolol that does not pass through the BBB was used as a reference for vascular contamination of brain tissue.

FIG. 3 shows the brain: plasma concentration ratio of the PEG-nalbuphine conjugate. The brain: plasma ratio of nalbuphine was 2.86: 1, indicating almost 3 times greater concentration of nalbuphine in the brain compared to the plasma compartment. PEG conjugation significantly reduced nalbuphine entry into the central nervous system, as evidenced by the relatively low brain: plasma ratio of PEG-nalbuphine conjugates. Conjugation with 3PEG units reduced the brain: plasma ratio to 0.23: 1, indicating that the concentration of 6-O-mPEG ₃ -nalbuphine in the brain was approximately 4 times lower than that in plasma. 6-O-mPEG ₆ -nalbuphine and 6-O-mPEG ₉ -nalbuphine were significantly excluded from the central nervous system because their brain: plasma ratio was not significantly different from the vascular marker atenolol. Become.

(Example 11)
(Preparation of oligomer-fentanyl conjugate)
mPEG _n -O-fentanyl conjugates can be prepared according to the procedure outlined below. Conventional organic synthesis techniques are used in performing this synthesis technique.

［式中、ｍＰＥＧ_ｎは、−（ＣＨ_２ＣＨ_２Ｏ）_ｎ−ＣＨ_３であり、かつｎは１〜９の整数である］
を調製するための例示的な手法が、下記に提供される。

The following structure, in which the PEG oligomer is located at the N- (1- (2-phenylethyl) piperidin-4-yl) phenyl group, ie covalently bonded:

[Wherein mPEG _n is — (CH ₂ CH ₂ O) _n —CH ₃ , and n is an integer of 1 to 9]
An exemplary approach for preparing is provided below.

In the above approach, the starting material is (haloethyl) hydroxybenzene, where the hydroxy group forms the point of attachment for the PEG oligomer. (Haloethyl) hydroxybenzene, or (bromoethyl) hydroxybenzene, reacts with a mesylated or halogenated activated mPEG oligomer, thereby forming the desired PEG-oligomer modified (haloethyl) benzene intermediate. This intermediate is then reacted with piperidin-4-one in the presence of a phase transfer catalyst and the bromo group is reacted at the piperidin-4-one nitrogen to yield the next intermediate 1- (mPEG _oligo -phenylethyl ) Piperidin-4-one is formed. The ketone functionality is then reduced in the presence of a reducing agent such as sodium borohydride and converted to an amino group, ie, N-phenyl-piperidin-4-amine, by reaction with aniline. Finally, the secondary amino group is converted to a tertiary amine by reaction with propionyl chloride to form the desired product as shown in the above scheme.

A subject mPEG _n -O-fentanyl conjugate having a PEG oligomer located at the N- (1- (2-phenylethyl) piperidin-4-yl) phenyl group is shown as shown in Scheme 11-B below. Was synthesized using a reaction scheme slightly modified from Scheme 11-A above.

The above approach uses tosyl (p-toluenesulfonate) leaving groups in various steps of the synthesis. The desired PEG-oligomer conjugate (n = 1-9) is polymerized by reacting ditosylated 3- (2-hydroxyethyl) phenol with N-phenyl-N- (piperidin-4-yl) propionamide. , N- (1- (3-hydroxyphenylethyl) piperidin-4-yl) -N-phenylpropionamide was formed in the tosylated form, followed by removal of the tosyl group. The PEG-oligomer group is then reacted at the phenyl hydroxyl position by reaction of N- (1- (3-hydroxyphenylethyl) piperidin-4-yl) -N-phenylpropionamide with a molar excess of mPEG _oligo -tosylate. Introduced into the molecule, the desired mPEG _n -O-fentanyl conjugate was formed. Commonly used reactant ratios and reaction conditions are provided in the above reaction scheme.

を提供するための例示的な手法を下記に示す。

The following structure in which the PEG oligomer is located at the N-phenyl group, ie covalently bonded:

An exemplary approach for providing is shown below.

The above exemplary procedure for forming mPEG _n -O-fentanyl conjugates with PEG oligomers located on the N-phenyl ring begins, for example, using 2-bromoethylbenzene as the starting material. 2-Bromoethylbenzene is reacted with piperidin-4-one in the presence of a phase transfer catalyst, thereby forming the resulting 1-phenethylpiperidin-4-one. 1-phenethylpiperidin-4-one was prepared by taking an N-protected hydroxyaniline and reacting it with an activated mPEG oligomer, such as bromomethoxy PEG _oligo or mPEG _oligo mesylate, followed by removal of the protecting group. Conjugate with mPEG _oligo- substituted aniline (see step (b) above). As shown in reaction step (c) above, 1-phenethylpiperidin-4-one is reacted with mPEG _oligo- substituted aniline in the presence of a reducing agent to convert the keto group to an amine, and the intermediate 1- forming a phenylethyl-4-ylamino -mPEG _oligo benzene. Finally, the secondary amino group is converted to a tertiary amine by reaction with propionyl chloride to form the desired product as shown in the above scheme.

Using a reaction scheme in which the subject mPEGn-O′-fentanyl conjugate having a PEG oligomer located at the N-phenyl group is slightly modified from Scheme 11-C above, as shown in Scheme 11-D below. And synthesized.

As shown in Scheme 11-D above, the desired mPEGn-O-fentanyl conjugate is first reacted with 1-phenethylpiperidin-4-one with 3-aminophenol under reducing conditions, thereby providing keto functionality. Was prepared by conversion to the amine, ie by reaction with the amino group of 3-aminophenol. The product 3- (1-phenethylpiperidin-4-ylamino) phenol is then reacted with N- (3-hydroxyphenyl) -N- (in the presence of a base (eg triethylamine) and dimethylaminopyridine (DMAP). 1-phenethylpiperidin-4-yl) propionamide was reacted with propionic anhydride under conditions effective to form. Finally, the introduction of oligomeric PEG functionality, and mPEG _oligo tosylate precursor N- (3- hydroxyphenyl)-N-(-4-1-phenethyl-yl) propionamide molar excess, the desired conjugate By reacting under conjugation conditions effective to form. In general, the ratio of reactants used and reaction conditions are provided in the above reaction scheme.

(Example 11A)
(Preparation of m-mPEG _n -O-fentanyl conjugate)
(Synthesis of m-mPEG ₁ -O-fentanyl conjugate (n = 1))

  The conjugate described above was prepared using the procedure shown in Example 11 and outlined in Scheme 11-B.

実施例１１に示され、スキーム１１−Ｂに概略的に記載された手法を使用して、上記の抱合体を調製した。 (Synthesis of m-mPEG ₂ -O-fentanyl conjugate (n = 2))

The above conjugate was prepared using the procedure shown in Example 11 and described schematically in Scheme 11-B.

実施例１１に示され、スキーム１１−Ｂに概略的に記載された手法を使用して、上記の抱合体を調製した。 (Synthesis of m-mPEG ₃ -O- fentanyl conjugate (n = 3))

The above conjugate was prepared using the procedure shown in Example 11 and described schematically in Scheme 11-B.

実施例１１に示され、スキーム１１−Ｂに概略的に記載された手法を使用して、上記の抱合体を調製した。 (Synthesis of m-mPEG ₄ -O-fentanyl conjugate (n = 4))

The above conjugate was prepared using the procedure shown in Example 11 and described schematically in Scheme 11-B.

実施例１１に示され、スキーム１１−Ｂに概略的に記載された手法を使用して、上記の抱合体を調製した。 (Synthesis of m-mPEG ₅ -O-fentanyl conjugate (n = 5))

The above conjugate was prepared using the procedure shown in Example 11 and described schematically in Scheme 11-B.

実施例１１に示され、スキーム１１−Ｂに概略的に記載された手法を使用して、上記の抱合体を調製した。 (Synthesis of m-mPEG ₆ -O-fentanyl conjugate (n = 6))

The above conjugate was prepared using the procedure shown in Example 11 and described schematically in Scheme 11-B.

実施例１１に示され、スキーム１１−Ｂに概略的に記載された手法を使用して、上記の抱合体を調製した。 (Synthesis of m-mPEG ₇ -O-fentanyl conjugate (n = 7))

The above conjugate was prepared using the procedure shown in Example 11 and described schematically in Scheme 11-B.

実施例１１に示され、スキーム１１−Ｂに概略的に記載された類似の手法を使用して、上記の抱合体を調製した。 (Synthesis of m-mPEG ₇ -O-fentanyl conjugate (n = 7))

The above conjugates were prepared using similar procedures shown in Example 11 and described schematically in Scheme 11-B.

実施例１１に示され、スキーム１１−Ｂに概略的に記載された手法を使用して、上記の抱合体を調製した。 (Synthesis of m-mPEG ₈ -O-fentanyl conjugate (n = 8))

The above conjugate was prepared using the procedure shown in Example 11 and described schematically in Scheme 11-B.

実施例１１に示され、スキーム１１−Ｂに概略的に記載された手法を使用して、上記の抱合体を調製した。 (Synthesis of m-mPEG ₉ -O-fentanyl conjugate (n = 9))

The above conjugate was prepared using the procedure shown in Example 11 and described schematically in Scheme 11-B.

Each of the above mPEG _1-9 -O-fentanyl conjugates was characterized by ¹ H NMR (200 MHz Bruker) and by LC / MS.

実施例１１に示され、スキーム１１−Ｄに概略的に記載された手法を使用して、上記の抱合体を調製した。このシリーズにおいて、オリゴマーｍＰＥＧをＮ−フェニル基のメタ位で共有結合させた。 (Example 12)
(Preparation of m-mPEG _n —O′-fentanyl conjugate)
(Synthesis of m-mPEG ₁ -O′-fentanyl conjugate (n = 1))

The conjugate described above was prepared using the procedure shown in Example 11 and outlined in Scheme 11-D. In this series, oligomeric mPEG was covalently attached at the meta position of the N-phenyl group.

上記の抱合体を、実施例１１に示され、スキーム１１−Ｄに概略的に記載された手法を使用して調製した。 (Synthesis of m-mPEG ₂ -O′-fentanyl conjugate (n = 2))

The above conjugate was prepared using the procedure shown in Example 11 and outlined in Scheme 11-D.

上記の抱合体を、実施例１１に示され、スキーム１１−Ｄに概略的に記載された手法を使用して調製した。 (Synthesis of m-mPEG ₃ -O'- fentanyl conjugate (n = 3))

The above conjugate was prepared using the procedure shown in Example 11 and outlined in Scheme 11-D.

上記の抱合体を、実施例１１に示され、スキーム１１−Ｄに概略的に記載された手法を使用して調製した。 (Synthesis of m-mPEG ₄ -O′-fentanyl conjugate (n = 4))

The above conjugate was prepared using the procedure shown in Example 11 and outlined in Scheme 11-D.

上記の抱合体を調製した。 (Synthesis of m-mPEG ₅ -O′-fentanyl conjugate (n = 5))

The above conjugate was prepared.

上記の抱合体を、実施例１１に示され、スキーム１１−Ｄに概略的に記載された手法を使用して調製した。 (Synthesis of m-mPEG ₆ -O′-fentanyl conjugate (n = 6))

The above conjugate was prepared using the procedure shown in Example 11 and outlined in Scheme 11-D.

上記の抱合体を、実施例１１に示され、スキーム１１−Ｄに概略的に記載された手法を使用して調製した。 (Synthesis of m-mPEG ₇ -O′-fentanyl conjugate (n = 7))

The above conjugate was prepared using the procedure shown in Example 11 and outlined in Scheme 11-D.

上記の抱合体を、実施例１１に示され、スキーム１１−Ｄに概略的に記載された手法を使用して調製した。 (Synthesis of m-mPEG ₈ -O′-fentanyl conjugate (n = 8))

The above conjugate was prepared using the procedure shown in Example 11 and outlined in Scheme 11-D.

上記の抱合体を、実施例１１に示され、スキーム１１−Ｄに概略的に記載された手法を使用して調製した。 (Synthesis of m-mPEG ₈ -O′-fentanyl conjugate (n = 8))

The above conjugate was prepared using the procedure shown in Example 11 and outlined in Scheme 11-D.

上記の抱合体を、実施例１１に示され、スキーム１１−Ｄに概略的に記載された手法を使用して調製した。 (Synthesis of m-mPEG ₉ -O′-fentanyl conjugate (n = 9))

The above conjugate was prepared using the procedure shown in Example 11 and outlined in Scheme 11-D.

Each of the above mPEG _1-9 -O′-fentanyl conjugates was characterized by ¹ H NMR (200 MHz Bruker) and by LC / MS.

(Example 13)
(Preparation of para-mPEG _n -O'-fentanyl conjugate)
(Synthesis of p-mPEG ₁ -O′-fentanyl conjugate (n = 1))

    The above conjugate can be prepared using the procedure shown in Example 11. In this series, oligomeric mPEG is covalently attached at the para position of the N-phenyl group.

(Synthesis of p-mPEG ₄ -O′-fentanyl conjugate (n = 4))

The para-substituted conjugate was prepared according to the reaction scheme shown below.

The desired pPEG ₄ -O-fentanyl conjugate is first reacted with 1-phenethylpiperidin-4-one with 4-aminophenol under reducing conditions (eg, in the presence of a reducing agent such as NaBH (OAc) ₃ ). And thereby converting the keto functionality to an amine, ie by reaction with the amino group of 4-aminophenol. The product 4- (1-phenethylpiperidin-4-ylamino) phenol is then reacted with N- (4-hydroxyphenyl) -N- in the presence of a base (eg, triethylamine) and dimethylaminopiiridine (DMAP). Reacted with propionic anhydride under conditions effective to form (1-phenethylpiperidin-4-yl) propionamide. Finally, the introduction of oligomeric PEG functionality to form the desired conjugate with the precursor N- (4-hydroxyphenyl) -N- (1-phenethylpiperidin-4-yl) propionamide and mPEG ₄ tosylate The reaction was carried out under effective conjugation conditions. In general, the ratio of reactants used and reaction conditions are provided in the above reaction scheme.

Additional pPEG _oligo- O-fentanyl conjugates can be similarly prepared.

(Example 14)
Preparation of mPEG _n -OM (mPEGn-O-mesylate) for use in Examples 15, 16, and 17.
In a 40 mL glass vial, mix HO—CH ₂ CH ₂ OCH ₂ CH ₂ —OH (1.2 mL, 10 mmol) and DIEA (N, N-diisopropylethylamine, 5.2 mL, 30 mmol, 3 eq), resulting in The resulting homogeneous colorless mixture was cooled to 0 ° C. and MsCl (1.55 mL, 20 mmol, 2 eq) was added slowly via a syringe over 4 minutes with vigorous stirring. Upon addition, a biphasic mixture resulted, with a yellow solid at the bottom and a clear supernatant. The ice bath was removed and the reaction was allowed to warm to room temperature overnight. At this point, the reaction was dissolved in water, extracted with CHCl ₃ (3 × 50 mL), washed with 2 × 50 m of a 0.1 M HCl / brine mixture followed by 50 mL of brine. The organic layer was dried over MgSO ₄ and filtered to give a yellow solution and evaporated to give a brown oil (2.14 g). ¹ H NMR confirms the identity of the product 3.3 (1H NMR δ3.1 (s, 3H), 3.3 (s, 3H), 3.5-3.55 (m, 2H), 3.6-3.65 (m, 2H), 3.7-3.75 (m, 2H), 4.3-4.35 (m, 2H) ppm).

All other PEG _n -OMS (n = 3, 4, 5, 6, 7, and 9) were prepared in a similar manner, and the final compound produced in each case was isolated as a brown oil It was. Mass spectrum and proton NMR data (not shown) confirmed the formation of the product PEGylated at the desired OM.

以下は、市販の硫酸モルヒネ水和物を使用する遊離塩基の調製（概して手順）を説明する。 (Example 15)
(Preparation of mPEG _n -O-morphine conjugate)

The following describes the preparation (generally a procedure) of the free base using commercially available morphine sulfate hydrate.

Spectrum morphine sulfate United States Pharmacopeia (510 mg) was dissolved in water (70 mL). The solution was then basified to pH 10 using aqueous K ₂ CO ₃ to give a white suspension. To the white suspension was added DCM (dichloromethane, 50 mL) but the solid could not be dissolved. The mixture was acidified with 1M HCl, resulting in a clear biphasic solution. The organic phase was separated off and the aqueous phase was carefully brought to pH 9.30 (monitored by pH meter) using the same K ₂ CO ₃ solution as above. A white suspension resulted again. The heterogeneous mixture was extracted with DCM (5 × 25 mL) and an insoluble white solid was incorporated into both the organic and aqueous layers. The organic layer was dried over MgSO ₄ , filtered and rotary evaporated to give 160 mg of morphine free base (56% recovery). Additional product was not recovered from the filter cake using MeOH, but an additional 100 mg was recovered from the aqueous phase by 2 × 50 mL extraction using EtOAc, giving a total yield of 260 mg (68% ).

  (MEM protection of morphine free base)

A general procedure for protecting the free base of morphine with the protecting group β-methoxyethoxymethyl ester (“MEM”) is shown schematically below.

Free base morphine (160 mg, 0.56 mmol) was dissolved in 20 mL acetone / toluene (2/1 mixture). To the resulting solution was added K ₂ CO ₃ (209 mg, 1.51 mmol, 2.7 equiv) followed by MEMCl (96 μL, 0.84 mmol, 1.5 equiv) and the resulting heterogeneous mixture was added. Stir overnight at room temperature. After 5 hours at room temperature, the reaction was deemed complete by LC-MS. Morphine free base retention time (standard 6 minutes, Onyx Monolyth C18 column, 50 × 4.6 mm; 0 with 0.1% TFA in water containing 0.1% TFA under standard 6 minute gradient operating conditions ~ 100% acetonitrile, 1.5 mL / min; detection: UV254, ELSD, MS; retention time is measured with a UV254 detector, ELSD has a delay of about 0.09 minutes, MS is With a delay of about 0.04 minutes) was 1.09 minutes; the retention time for the product was 1.54 minutes (standard 6 minutes) and the main impurity was 1.79 minutes . The reaction mixture was evaporated to dryness, dissolved in water and extracted with EtOAc (3 times, the combined organic layers were washed with brine, dried over MgSO ₄ , filtered and rotary evaporated), 160 mg (77 %) Of the desired product as a colorless oil. The purity of the product was estimated to be about 80% by UV254.

  (Direct MEM protection of morphine sulfate (general procedure))

A general procedure for protecting morphine sulfate with the protecting group β-methoxyethoxymethyl ester (“MEM”) is shown schematically below. Although not explicitly shown in the scheme below, morphine is actually morphine sulfate hydrate, morphine. 0.5 H ₂ SO ₄ . 2.5H ₂ O.

To a suspension of 103 mg morphine sulfate hydrate (0.26 mmol) in 10 mL of 2: 1 acetone: toluene solvent mixture, 135 mg (1 mmol, 3.7 eq) K ₂ CO ₃ was added and suspended. The suspension was stirred at room temperature for 25 minutes. To the resulting suspension 60 μL (0.52 mmol) MEMCl was added and the mixture was allowed to react at room temperature. The mixture was added after 1 hour (38% nominal conversion, additional peak at 1.69 minutes and 2.28 minutes), after 3 hours (40% nominal conversion, additional at 1.72 minutes) Peak (M + 1 = 493.2)), after 4.5 hours (56% nominal conversion, additional peak at 1.73 minutes), and after 23 hours (> 99% nominal conversion, 1. An additional peak at 79 minutes-about 23% of the product peak by height at UV ₂₅₄ ), after which the reaction was quenched with MeOH, evaporated, extracted with EtOAc, and 160 mg of clear oil Got.

The same reaction was performed on 2 g (5.3 mmol) morphine sulfate hydrate, 2.2 g (16 mmol, 3 eq) K ₂ CO ₃ , 1.2 mL (10.5 mmol, 2 eq) in 100 mL solvent mixture. Repeated starting with MEMCl. Sampling after 2 hours (61% nominal conversion, 1.72 min surplus peak (M + 1 = 492.8)), 1 day after (80% nominal conversion, 1.73 min surplus Peak)) after 3 days (85% nominal conversion, only a small amount of impurities, 12 min gradient run) and after 6 days (91% conversion), after which the reaction was stopped, evaporated and EtOAc And purified on a combiflash using a 40 g column, DCM: MeOH 0-30% mobile phase. Three peaks were identified (instead of two) where an intermediate peak, 1.15 g (58% yield) of a light yellow oil, UV254 purity about 87% was recovered.

  (Conjugation of MEM protected morphine to provide MEM protected morphine conjugate)

A general procedure for conjugating MEM protected morphine with a water soluble oligomer to provide a MEM protected morphine PEG-oligomer conjugate is outlined below.

  Toluene / DMF solution (2: 1 mixture, 10 volumes total) is added to MEM-morphine free base followed by NaH (4-6 eq) followed by pre-prepared PEGnOM (1.2-1. 4 equivalents). The reaction mixture was heated to 55-75 ° C. and stirred until reaction completion was confirmed by LC-MS analysis (12-40 hours depending on PEG chain length). The reaction mixture was quenched with methanol (5 volumes) and the reaction mixture was evaporated to dryness under vacuum. The residue was redissolved in methanol (3 volumes) and chromatographed using a Combiflash system (0-40% MeOH / DCM). Fractions containing a large amount of product were collected, combined and evaporated to dryness. This material was then purified by reverse phase HPLC to give the product as a yellow to orange oil.

  (Deprotection of MEM protected morphine conjugates to provide morphine conjugates)

A general procedure for deprotecting a MEM protected morphine conjugate to provide a morphine conjugate is outlined below.

A solution of MEM protected morphine conjugate TFA salt suspended in DCM (8 volumes) was loaded with 6 volumes of 2M HCl in diethyl ether. The reaction mixture was allowed to stir at room temperature for 2 hours and then evaporated to dryness under reduced pressure. The oily residue was dissolved in MeOH (8 vol), filtered through glass wool and evaporated under reduced pressure to give a viscous orange to yellow oil in quantitative yield. Compounds produced by this method include α-6-mPEG ₃ -O-morphine (Compound A, n = 3), 217 mg HCl salt, 97% purity (95% by UV254; 98% by ELSD); -6-mPEG ₄ -O- morphine (compound a, n = 4), HCl salt of 275 mg, 98% purity (97% by UV254; 98% by ELSD); α-6-mPEG 5 -O- morphine (compound a, n = 5), HCl salt of 177 mg, 93% by 95% purity (UV254; 98% by ELSD); α-6-mPEG 6 -0- morphine (compound a, n = 6), HCl salt of 310mg , 98% pure (98 by UV254; 99% by ELSD); α-6-mPEG 7 -O- morphine (compound a, n = 7), HCl salt of 541 mg, 96% pure (93% by UV254; by ELSD 99%); and _{α-6-mPEG-O 9} - morphine (Compound A, n = 9), HCl salt of 466 mg, 97% by 98% purity (UV254; 99% by ELSD )including. In addition, a single PEG monomer linked morphine conjugate, α-6-mPEG ₁ -O-morphine (Compound A, n = 1), 124 mg HCl salt, 97% purity (95% purity by UV ₂₅₄ ; 98% by ELSD), and α-6-mPEG2-O-morphine (Compound A, n = 2), 485 mg HCl salt, 97% purity (95% purity by UV ₂₅₄ ; 98% purity by ELSD) as well Prepared.

(Example 16)
(Preparation of mPEGn-O-codeine conjugate)

A general procedure for conjugating codeine with an activated sulfonated ester of a water-soluble oligomer to provide codeine conjugates (using mPEG ₃ OMs as a representative oligomer) is outlined below.

Codeine (30 mg, 0.1 mmol) was dissolved in a toluene / DMF (75: 1) solvent mixture followed by HO—CH ₂ CH ₂ OCH ₂ CH ₂ OCH ₂ CH ₂ OMs (44 mL, 2 eq) and NaH (in mineral oil). 60% suspension, 24 mg, 6 eq) was added. The resulting homogeneous yellow solution was heated to 45 ° C. After 1 hour, the reaction showed 11% conversion (2.71 min surplus peak, 12 minute trial), and after 8 hours the reaction showed 7% conversion (3.30 min surplus peak, 12 min). After 24 hours, the reaction showed a conversion of 24% (the extent of the excess peak is 1.11 minutes and 2.79 minutes with the two highest ones). At this point, an additional 16 mg of NaH was added and heating was continued for 6 hours, followed by an additional 16 mg of NaH, followed by heating for 66 hours. Thereafter, no starting material remained and analysis revealed many extra peaks, with the two highest corresponding to 2.79 min and 3 min (the product peak was at least 7 peaks The second highest of them).

This synthesis was repeated using a 10-fold scale, where 30 mL of solvent mixture was used. After 18 hours, analysis revealed a 71% nominal conversion with an additional peak in UV (one high peak is 3.17 minutes, many small peaks, where the desired peak is (Corresponds to 3.43 minutes in UV). Then, after adding 80 mg (2 mmol) of NaH, heating was continued. After 3 hours, analysis revealed a nominal conversion of 85% (several peaks, 3.17 minutes major). The reaction mixture was diluted with water and extracted with EtOAc (3 times, the combined organic layers were washed with brine, dried over MgSO ₄ , filtered and rotary evaporated) to give a yellow oil (LC -MS had no starting material, 90% purity by ELSD, 50% purity by UV-major impurities were at 3.2 minutes). The crude product was dissolved in DCM, applied to a small cartridge filled with SiO ₂ of 230-400 mesh, dried at Combi- flash through the column cartridge packed pre-4g, solvent A = DCM and the solvent Elution using B = MeOH, gradient 0-30% B. The analysis revealed two peaks that were hardly symmetrical: a small leading peak and a larger peak with a tail. Fractions were analyzed using LC-MS where none were identified as containing pure product. Combined fractions containing either product (tt # 22-30) were 150 mg (34% yield) of impure product (LC-MS purity at 3.35 min by UV254, after solvent evaporation, Here, 25% produced the major impurity, which represented 3.11 minutes, 3.92 minutes, 4.32 minutes, 5.61 minutes by UV254 in a 12 minute trial). A second purification by HPLC using a gradient corresponding to 15-60% B, 70 min, 10 mL / min (solvent A = water, 0.1% TFA; solvent B = acetonitrile, 0.1% TFA) was As a result, the adjacent peaks were hardly separated. Only two fractions were clean enough to give 21 mg of TFA (> 95% purity, 4.7% yield). Both three additional fractions before and after the fraction containing the desired product (for a total of six additional fractions) were combined to give 70 mg of approximately 50% pure product as a TFA salt.

  Using this same approach, other conjugates with different numbers of ethylene oxide units (n = 4, 5, 6, 7, and 9) were generated using these NaH conditions outlined above. .

  Conversion of codeine-oligomer conjugate TFA salt to codeine-oligomer HCl salt

The general procedure for converting codeine-oligomer TFA salt to codeine-oligomer HCl salt is outlined below.

A solution of codeine-oligomer conjugate TFA salt suspended in DCM (8 volumes) was loaded with 6 volumes of 2M HCl in diethyl ether. The reaction mixture was allowed to stir at room temperature for 2 hours and then evaporated to dryness under reduced pressure. The oily residue was dissolved in MeOH (8 vol), filtered through glass wool and evaporated under reduced pressure to give a viscous orange to yellow oil in quantitative yield. After this general procedure, the following compounds: α-6-mPEG ₃ -O-codeine (Compound B, n = 3) 235 mg HCl salt, 98% purity; α-6-mPEG ₄ -O-codeine (Compound B, n = 4) 524 mg HCl salt, 98% purity; α-6-mPEG ₅ -O-codeine (Compound B, n = 5) 185 mg HCl salt, 98% purity +119 mg HCl salt 97% purity , Α-6-mPEG ₆ -O-codeine (Compound B, n = 6) 214 mg HCl salt, 97% purity; α-6-mPEG ₇ -O-codeine (Compound B, n = 7) 182 mg HCl salt , 98% purity; α-6-mPEG ₉ -O-codeine (Compound B, n = 9) 221 mg HCl salt, 97% purity; α-6-mPEG ₁ -O-codeine (Compound B, n = 1) 63 mg HCl salt , 90% purity; and α-6-mPEG ₂ -O-codeine (Compound B, n = 2) 178 mg HCl salt, 90% purity was synthesized.

（「ｍＰＥＧ_ｎＯＭｓ」を代表的なオリゴマーとして使用して）ヒドロキシコドンを水溶性オリゴマーの活性化型スルホン酸エステルと抱合してヒドロキシコドン抱合体を提供するための一般的な手法を下に概略的に示す。

(Example 17)
(Preparation of mPEG _n -O-hydroxycodone conjugate)

The general procedure for conjugating hydroxycodone with an activated sulfonate ester of a water soluble oligomer to provide a hydroxycodone conjugate (using “mPEG _n OMs” as a representative oligomer) is outlined below. Indicate.

(Reduction of oxycodone to α-6-hydroxycodone)
To a solution of oxycodone free base in anhydrous THF cooled to −20 ° C. under nitrogen was added a 1.0 M THF solution of potassium tri-sec-butylborohydride over 15 minutes. The solution was stirred at −20 ° C. under nitrogen for 1.5 hours before water (10 mL) was added slowly. The reaction mixture was stirred for an additional 10 minutes at −20 ° C. and then allowed to warm to room temperature. All solvent was removed under reduced pressure and CH ₂ Cl ₂ was added to the remaining residue. The CH ₂ Cl ₂ phase was extracted with 0.1 N HCl / NaCl aqueous solution and the combined 0.1 N HCl solution extracts were washed with CH ₂ Cl ₂ , then Na ₂ CO ₃ was added, pH = 8 Adjusted. The solution was extracted with CH ₂ Cl ₂ . The CH ₂ Cl ₂ extract was dried over anhydrous Na ₂ SO ₄ . After removing the solvent under reduced pressure, the desired α-6-HO-3-hydroxycodone was obtained.

(Conjugation of mPEG _n OMs to α-6-hydroxycodone)
Toluene / DMF solution (2: 1 mixture, total 10 volumes) was added to hydroxycodone (prepared as indicated in the previous section) followed by NaH (4 eq) and then mPEG _n OMs (1.3 eq). ) Was loaded. The reaction mixture was heated to 60-80 ° C. and stirred until reaction completion was confirmed by LC-MS analysis (12-40 hours depending on PEG chain length). The reaction mixture was quenched with methanol (5 volumes) and the reaction mixture was evaporated to dryness under vacuum. The residue was redissolved in methanol (3 volumes) and chromatographed using Combiflash (0-40% MeOH / DCM). Fractions containing a large amount of product were collected, combined and evaporated to dryness. This material was then purified by reverse phase HPLC to provide the final product as a yellow to orange oil.

(Conversion of hydroxycodone conjugate TFA salt to hydroxycodone conjugate HCl salt)
A solution of hydroxycodone conjugate TFA salt suspended in DCM (8 volumes) was loaded with 6 volumes of 2M HCl in diethyl ether. The reaction mixture was allowed to stir at room temperature for 2 hours and then evaporated to dryness under reduced pressure. The oily residue was dissolved in MeOH (8 vol), filtered through glass wool and evaporated under reduced pressure to give a viscous orange to yellow oil in quantitative yield. After this general procedure, 242 mg HCl salt of the following compound: α-6-mPEG ₃ -O-oxycodone (also known as α-6-mPEG ₃ -O-hydroxycodone) (Compound C, n = 3), 96% purity; α-6-mPEG ₄ -O-oxycodone (also known as α-6-mPEG ₄ -O-hydroxycodone) (Compound C, n = 4) 776 mg HCl salt, 94% purity; α-6-mPEG _5- O-oxycodone (also known as α-6-mPEG ₅ -O-hydroxycodone) (Compound C, n = 5) 172 mg HCl salt, 93% purity; α-6-mPEG ₆ -O-oxycodone (also known as α- 6-mPEG ₆ -O-hydroxycodone) (Compound C, n = 6) 557 mg HCl salt, 98% purity; α-6-mPEG ₇ -O-oxycodone (also known as α-6-mPEG ₇ -O) -Hydroxycodone) (compound C, n = 7) 695 mg HCl salt, 94% purity; and α-6-mPEG ₉ -O-oxycodone (also known as α-6-mPEG ₉ -O-hydroxycodone) (compound C, n = 9) 435 mg of HCl salt 95% purity was synthesized. Α-6-mPEG ₁ -O-oxycodone (also known as α-6-mPEG ₁ -O-hydroxycodone) (Compound C, n = 1) 431 mg HCl salt 99% purity; and α-6 -MPEG ₂ -O-oxycodone (also known as α-6-mPEG ₂ -O-hydroxycodone) (Compound C, n = 2) 454 mg of HCl salt, 98% purity was prepared similarly.

(Example 18)
In Vivo Analgesia Assay: _{Phenylquinone Distress} Using the analgesia assay, the following conjugate series: mPEG _2-7,9- O-morphine, mPEG _3-7,9- O-codeine, and mPEG _{1-4,6 It} was determined whether an exemplary PEG-oligomer-opioid agonist conjugate belonging to _{, 7,9-} O-hydroxycodone is effective in reducing and / or preventing visceral pain in mice.

  The assay utilized CD-1 male mice (5-8 individuals per group), each mouse approximately 0.020-0.030 kg on the day of the study. Mice were treated according to standard protocols. A compound that lacks the covalent bond of a water-soluble non-peptide oligomer to a mouse (ie, a parent molecule that has not been modified with a PEG oligomer), a corresponding version that contains the compound covalently attached to a water-soluble non-peptide oligomer (ie, a conjugate) Or a single “pre-treatment” dose of a control solution (intravenous, subcutaneous, intraperitoneal or orally) was given 30 minutes prior to administration of the phenylquinone (PQ) solution. Each animal was given an intraperitoneal injection of an irritant (phenylquinone, PQ) that induced "bitterness" that could include abdominal contractions, torso twisting and turning, dorsal arch formation, and hindlimb extension. Each animal received an intravenous injection of PQ (1 mg / kg PQ, 0.1 mL / body weight 10 g). After injection, the animals were returned to their observation cages and their behavior was observed. Shrinkage was counted 35-45 minutes after “pretreatment”. Animals were used a single time. Each substance tested was administered in the range of 0.1-100 mg / kg (n = 5-10 individuals / dose).

The results are shown in FIG. 4 (mPEG _2-7,9- O-morphine and control), FIG. 5 (mPEG _1-4,6,7,9- O-hydroxycodone and control), and FIG. 6 (mPEG _{3-7 , 9-} O-codeine and control). ED ₅₀ values are provided in Table 3A and Table 3B below.

(Example 19)
(In vivo analgesia assay: hot plate latency assay)
Examples belonging to the following conjugate series using the hot plate latency analgesia assay: mPEG _1-5 -O-morphine, mPEG1-5-O-hydroxycodone, and mPEG _2-5,9- O-codeine It was determined whether a typical PEG-oligomer-opioid agonist conjugate would be effective in reducing and / or preventing visceral pain in mice.

  The assay utilized CD-1 male mice (10 individuals per group), each mouse approximately 0.028-0.031 kg on the day of the study. Mice were treated according to standard protocols. A compound that lacks a covalent bond of a water-soluble non-peptide oligomer (ie, an unmodified parent molecule), a corresponding version containing the compound covalently attached to a water-soluble non-peptide oligomer (ie, a conjugate), or a control A single “pre-treatment” dose of solution (subcutaneous) was given 30 minutes prior to the hot plate test. The hot plate temperature was set at 55 ± 1 ° C. and calibrated using a surface thermometer before the start of the experiment. Thirty minutes after “pre-treatment”, each mouse was placed on a hot plate and the latency to lick the hind legs was recorded in units of 0.1 seconds. Mice were removed if they did not lick within 30 seconds. Immediately following the hot plate test, a temperature probe was inserted 17 mm into the rectum and body temperature was read in units of 0.1 ° C. when the thermometer was stable (approximately 10 seconds). Animals were used a single time. Each substance tested was administered in the range of 0.3-30 mg / kg (n = 5-10 individuals / dose).

  The results are shown in FIG. 7 (hydroxycodone series), FIG. 8 (morphine series), and FIG. 9 (codeine). The plot shows latency (time to lick hind legs (seconds)) and dose of compound administered (mg / kg).

(Example 20)
Pharmacokinetics of PEG _oligo -opioid compounds after intravenous (IV) and oral (PO) administration in male Sprague-Dawley rats-study design
175 male Sprague-Dawley rats (Charles River Labs, Hollister, Calif.) With an indwelling jugular vein catheter and an indwelling carotid artery catheter (JVC / CAC) were utilized in this study. 3 individuals / group. All animals were fasted overnight. Prior to dosing, the rats were weighed, the tail and cage card were labeled for identification, and the dose was calculated. Anesthesia was induced and maintained with 3.0-5.0% isoflurane. JVC and CAC were exposed externally, flushed with HEP / saline (10 IU / mL HEP / ml saline), capped and labeled to identify the jugular vein and carotid artery. Predose samples were collected from JVC. When all animals have recovered from anesthesia and the pre-dose sample has been processed, animals for the intravenous group are administered intravenously (IV) via JVC using a 1 mL syringe containing the appropriate test substance. The catheter dead space was flushed with 0.9% saline to ensure that the animals received the correct dose, and the oral group animals were treated orally via gavage.

  After a single IV administration, blood samples are collected at 0 (before administration as collected above), 2, 10, 30, 60, 90, 120, and 240 minutes, and after oral administration, blood samples are collected at 0 (above Collected via the carotid catheter at 15, 30, 60, 120, 240, and 480 minutes and processed as described in the protocol. Animals were euthanized after the last harvest.

  Bioanalytical analysis of plasma samples was performed using LC-MS / MS method.

(Pharmacokinetic analysis)
Pharmacokinetic analysis was performed using WinNonlin (version 5.2, Mountain View, CA-94014), replacing plasma concentrations that were below the lower limit of quantification (LLOQ) with 0 before generating the table and pharmacokinetic analysis It was. The following pharmacokinetic parameters were estimated using the plasma concentration-time characteristics of each animal.
C ₀ concentration extrapolated to time “0” C _max maximum (peak) concentration AUC _all 0 to the time of the last concentration value area under time T _{1/2 (Z)} end removal half-life AUC _inf Concentration from 0 to infinite time-area under time T _max maximum or peak concentration after administration CL systemic clearance V size of distribution based on _z- terminal phase V _ss size of steady state distribution MRT _last Average residence time to the last observable concentration F Bioavailability

  Oral bioavailability is estimated using AUCall data normalized by the average dose for the compound, where one of group IV or PO is for n = 3 / group Only data were reported.

(Example 21)
(Pharmacokinetics of mPEGn-O-hydroxycodone conjugate at IV and PO)
Pharmacokinetic studies were performed in Sprague-Dawley rats as described in Example 20 above. The compounds administered were mPEG _n- O-hydroxycodone conjugate (where n = 1, 2, 3, 4, 5, 6, 7, and 9) and the parent compound oxycodone. The objective was to determine the pharmacokinetics of the parent compound and its various oligomer conjugates administered both intravenously and orally.

Oxycodone, mPEG ₀ -O- oxycodone, mPEG ₁ -O- hydroxy codon, mPEG ₂ -O- hydroxy codon, mPEG ₃ -O- hydroxy codon, mPEG ₄ -O- hydroxy codon, mPEG ₅ -O- hydroxy codon, mPEG Summary of plasma pharmacokinetic parameters after delivery with IV (1 mg / kg) and PO (5 mg / kg) for _6- O-hydroxycodone, mPEG ₇ -O-hydroxycodone, and mPEG ₉ -O-hydroxycodone. It is shown in the following tables: Table 4 and Table 5.

Based on the data observed for IV administration (Table 4), mPEG ₉ -O-hydroxycodone averages three times higher than the corresponding average t _1/2 value plasma concentration observed after parent oxycodone was given. It appeared that a plasma concentration of t _1/2 was reached.

  FIG. 10 shows the mean plasma concentration-time characteristics for mPEGn-O-hydroxycodone compounds administered IV as described above and for oxycodone itself when administered at a concentration of 1.0 mg / kg.

Based on the data observed for oral administration (Table 5), mPEG ₅ -O-hydroxycodone, mPEG ₆ -O-hydroxycodone, and mPEG ₇ -O-hydroxycodone were compared to the parent molecule oxycodone in plasma. Among them, a higher average exposure (approximately 3-8 times) appeared to be reached.

FIG. 11 shows the mean plasma concentration-time characteristics for the above mPEG _n -O-hydroxycodone compounds and for oxycodone when orally administered to rats at a concentration of 5.0 mg / kg.

  To summarize the results, intravenous administration of PEGylated hydroxycodone with different oligomeric PEG lengths (PEG1-PEG9) resulted in different plasma concentrations and exposure compared to oxycodone. PEGs with chain lengths 3, 5, 7, and 9 showed higher average exposure (AUC), whereas PEG6 showed equivalent average exposure (AUC) and chain lengths 1, 2, or 4 Having PEG showed slightly lower average exposure (AUC). Compounds with a PEG chain greater than 5 showed a tendency for lower clearance, higher distribution size at steady state, and increased removal half-value with increasing PEG chain length.

  Oral administration of PEGylated hydroxycodone with various oligomeric PEG lengths (PEG1-PEG9) resulted in improvements in plasma exposure, with the exception of hydroxycodone covalently attached to PEG1 and PEG3. Oral bioavailability is greatest for hydroxycodone covalently attached to PEG6 (55.3%), followed by 37.6% and 28.m for mPEG5-hydroxycodone and mPEG7-hydroxycodone, respectively. 1%. The elimination half-value tended to increase with increasing PEG chain.

(Example 22)
(Pharmacokinetics of mPEG _n -O-morphine conjugate at IV and PO)
Pharmacokinetic studies were performed in Sprague-Dawley rats as described in Example 20 above. The compounds administered were mPEG _n- O-morphine conjugates (where n = 1, 2, 3, 4, 5, 6, 7, and 9) and the parent compound morphine. The objective was to determine the pharmacokinetics of the parent compound and its various oligomer conjugates administered both intravenously and orally.

Morphine, mPEG ₁ -O-morphine, mPEG ₂ -O-morphine, mPEG ₃ -O-morphine, mPEG ₄ -O-morphine, mPEG ₅ -O-morphine, mPEG ₆ -O-morphine, mPEG ₇ -O-morphine Table 6 and Table 7 summarize the plasma pharmacokinetic parameters after the IV (1 mg / kg) and PO (5 mg / kg) routes for mPEG ₉ -O-morphine, respectively.

For the intravenous group, FIG. 12 shows the mean plasma concentration-time characteristics for mPEG _n -O-morphine described above after 1.0 mg / kg intravenous administration to rats. mPEG 2 _-O- do not match the plasma profile of morphine, appeared to be one outlier data in each animal was excluded from the pharmacokinetic analysis.

Based on the observed data (Table 6), mPEG ₉ -O-morphine is an average t _1/2 value four times higher than the corresponding t _1/2 value plasma concentration observed after parental morphine was given. Seemed to reach a plasma concentration of.

For the oral group, FIG. 13 shows the mean plasma concentration-time characteristics for the mPEG _n -O-morphine conjugate described above after oral administration to rats (5.0 mg / kg).

Based on the observed data (Table 7), mPEG ₄ -O-morphine appeared to reach the maximum plasma concentration of the conjugates tested compared to the parent molecule morphine.

  In summary, for IV data, administration of oligomeric pegylated morphine with various PEG lengths (PEG1-PEG9) resulted in higher plasma concentrations and exposure (AUC) compared to morphine itself. After 5 PEG there was a clear trend of increasing average AUC with increasing PEG chain, with a 10-fold higher average AUC for PEG9-morphine compared to unconjugated morphine. Also, the average half-life and average residence time increased with increasing PEG length. The lower average clearance value was consistent with the higher average AUC value observed.

The average size of the distribution estimated for the steady state immediately decreased by a factor of 5 with the introduction of a single PEG and reached a steady value at a PEG length of 5. Overall, PEGylation appeared to prolong removal t _1/2 and reduce morphine tissue distribution.

  Based on oral data, administration of PEGylated morphine conjugates with various PEG lengths (PEG1-PEG9) resulted in reduced oral bioavailability compared to morphine. The decrease in bioavailability appeared to be associated with the absorbing element rather than the metabolic element for these PEG-conjugates. Of the PEG-conjugates, those with a PEG length of 4 showed the greatest F value (22.1%), whereas those with shorter or longer PEG lengths lead to loss of absorption. Showed an obvious trend.

  In this study, the F% value for morphine was 3 times higher than the literature value of 15% at 7.5 mg / kg (J. Pharmacokinet. Biopharm. 1978, 6: 505-19). The reason for this higher exposure is unknown.

(Example 23)
(Pharmacokinetics of mPEG _n -O-codeine conjugate at IV and PO)
Pharmacokinetic studies were performed in Sprague-Dawley rats as described in Example 20 above. The compounds were mPEG _n -O-codeine conjugates (n = 1, 2, 3, 4, 5, 6, 7, and 9) and the parent compound codeine (n = 0). The goal was to determine the pharmacokinetics of the parent compound, codeine, and its various oligomer conjugates, administered both intravenously and orally.

Codeine, mPEG ₁ -O- codeine, mPEG ₂ -O- codeine, mPEG ₃ -O- codeine, mPEG ₄ -O- codeine, mPEG ₅ -O- codeine, mPEG ₆ -O- codeine, mPEG ₇ -O- codeine A summary of plasma pharmacokinetic parameters after IV (1 mg / kg) and PO (5 mg / kg) for mPEG ₉ -O-codeine is shown in Table 8 and Table 9, respectively.

For group IV, FIG. 14 shows the mean plasma concentration-time characteristics for the parent molecular codeine after intravenous administration and for the mPEG _n -O-codeine conjugate described above.

Based on the observed data (Table 8), mPEG ₆ -O-codeine is approximately 2.% of the plasma concentration of the corresponding t _1/2 value observed after administration of the parent molecule codeine among the conjugates tested. It seemed to reach a higher plasma concentration with a five-fold average t _1/2 value.

For oral group, 15, the average plasma concentration of the parent molecule codeine pair mPEG _n -O- codeine after oral administration to rats (5.0mg / kg) - shows the time characteristics.

Based on the observed data (Table 9), the PEG-6 compound, mPEG ₆ -O-codeine, like the parent molecule codeine, had the highest plasma concentration of the tested conjugates (mean AUCall 52 Twice as high).

In summary, for IV data, PEGylation of codeine with various oligomeric PEG lengths (PEG1 to PEG9) improved exposure (average AUC) only slightly, with moderate improvement (approximately 4 times) PEG- Observed for 6 conjugates. The size of the clearance and distribution was reduced 4-fold for this PEG-conjugate. Conjugates with PEG lengths 7 and 9 showed longer average t1 _{/ 2} values, but the average clearance and mean size of distribution (Vss) were determined for PEG7-codeine conjugates and PEG9-codeine conjugates. Decreased for both.

For oral data, oral bioavailability for codeine is very low (F = 0.52%). Oral bioavailability appears to increase with increasing PEG length after 2PEG, reaching a maximum at 32% bioavailability for codeine conjugates with PEG length 6. Then declined. In general, average t _1/2 and average residence values increased with PEG length. Of all the compounds tested, there was no difference in the time to reach the peak concentration (Tmax = 15), suggesting that the absorption was rapid and the absorption rate did not change.

(Example 24)
(In vitro binding of mPEG _n -O-opioid conjugate to opioid receptor)
The binding affinity of various PEG-opioid conjugates (mPEG _n -O-morphine, mPEG _n -O-codeine, and mPEG _n -O-hydroxycodone) was compared to cloned human mu opioid receptors, kappa opioid receptors. Or measured in vitro in membrane preparations prepared from CHO cells that heterologously express the delta opioid receptor. Radioligand displacement was measured using a scintillation proximity assay (SPA).

Briefly, serial dilutions of test compounds were placed in 96-well plates to which SPA beads, membranes, and radioligand were added. The assay conditions for each opioid receptor subtype are listed in Table 10 below. Plates were incubated at room temperature for 8 hours to overnight, centrifuged at 1000 rpm to pellet SPA beads, and radioactivity was measured using a TopCount® microplate scintillation counter. Specific binding at each concentration of test compound was calculated by subtracting non-specific binding measured in the presence of excess non-radioactive ligand. IC ₅₀ values were obtained by non-linear regression between specific binding and concentration curves, and Ki values were calculated using experimentally predetermined Kd values for each lot of membrane preparation.

The binding affinities of oligomeric PEG conjugates of morphine, codeine, and hydroxycodone are shown in Table 11. Overall, all conjugates showed measurable binding to mu opioid receptors, consistent with known pharmacological effects on the parent molecule. For a given PEG size, the order of increasing mu opioid binding affinity was PEG-morphine>PEG-hydroxycodone> PEG-codeine. The increase in PEG size resulted in a gradual decrease in the binding affinity of all PEG conjugates to the mu opioid receptor compared to the unconjugated parent molecule. However, PEG-morphine conjugates still retained high binding affinity and were within 15 times the binding affinity of the parent morphine. The mu opioid binding affinity of PEG-hydroxycodone was 20-50 times lower than the mu opioid binding affinity of PEG-morphine conjugates. Codeine and its PEG conjugate bound with very low affinity to the mu opioid receptor. Also, PEG-morphine conjugates bound to kappa opioid receptors and delta opioid receptors, and the order of selectivity was mu>kappa> delta. The binding affinity of codeine conjugates and hydroxycodone conjugates to kappa opioid receptors and delta opioid receptors was significantly lower than that of mu opioid receptors.

  Not applicable indicates that a Ki value could not be calculated because 50% inhibition of binding was not achieved at the maximum concentration of compound tested.

(Example 25)
(Efficacy in vitro mPEG _n -O- opioid conjugates to inhibit cAMP formation)
The effectiveness of various PEG-opioid conjugates was measured by their ability to inhibit cAMP formation after receptor activation. Studies were performed in CHO cells that heterologously express cloned human mu opioid receptors, kappa opioid receptors, or delta opioid receptors. cAMP was measured using the cAMP HiRange homogeneous time-resolved fluorescence assay (HTRF assay) based on the competitive immunoassay principle (Cisbio, catalog number 62AM6PEC).

Briefly, suspensions of cells expressing either mu opioid receptor, kappa opioid receptor, or delta opioid receptor were prepared in a buffer containing 0.5 mM isobutyl-methylxanthine (IBMX). . The cells were incubated with various concentrations of PEG-opioid conjugate and 3 μM forskolin for 30 minutes at room temperature. cAMP was detected according to the manufacturer's instructions according to the two-step assay protocol and time-resolved fluorescence was measured with the following settings: 330 nm excitation; 620 nm and 665 nm emission; 380 nm dichroic mirror. The 665 nm / 620 nm ratio is expressed as Delta F% and the test compound related data is expressed as a percentage of the mean maximum response in wells without forskolin. The EC ₅₀ was calculated for each compound from a sigmoidal dose response plot of concentration versus maximum response. In order to determine whether a compound behaves as a full or partial agonist in the system, the maximum response at the maximum concentration tested of the compound was compared to the maximum response produced by a known full agonist.

EC ₅₀ values for inhibition of cAMP formation in whole cells are shown in Table 12. Morphine, codeine, and oligomeric PEG conjugates of hydroxycodone were full agonists of the mu opioid receptor, despite having varying efficacy. Morphine and its conjugates were the most potent of the three series of opioids tested, whereas PEG hydroxycodone conjugates and PEG codeine conjugates showed significantly lower efficacy. A gradual decrease in the effectiveness of the PEG-morphine conjugate was observed with increasing PEG size, but the conjugate retained muon agonist potency within 40-fold that of the parent. In contrast to effects at the mu opioid receptor, morphine and PEG-morphine conjugates behaved as weak partial agonists at the kappa opioid receptor, yielding 47-87% of the maximum possible response. EC ₅₀ values were calculated for codeine and hydroxycodone conjugates at the kappa and delta opioid receptors since a complete dose response curve could not be generated over the range of concentrations tested (up to 500 μM). I couldn't.

Overall, the results of receptor binding and functional activity indicate that PEG-opioids are mu agonists in vitro.

(Example 26)
(Brain: plasma ratio of mPEG _n -O-opioid conjugate)
The ability of oligomeric mPEG-O-morphine conjugates, mPEG-O-codeine conjugates, and mPEG-O-hydroxycodone conjugates to cross the blood brain barrier (BBB) and enter the CNS (central nervous system) Later it was evaluated by measuring the brain: plasma ratio in rats.

Briefly, groups of 3 rats are injected intravenously (iv) with 5 mg / kg morphine, mPEG _n -O-morphine conjugate, codeine, and m-PEG _n -O-codeine conjugate each. did. PEG-2-oxycodone conjugate, PEG-3-oxycodone conjugate, and PEG-4-oxycodone conjugate are administered intravenously at 10 mg / kg, and oxycodone and other PEG-sized oxycodone conjugates are intravenously administered at 1 mg / kg. It was administered internally. The dose of oxycodone conjugate had to be adjusted to allow detection of sufficient concentrations in brain tissue. Atenolol, which does not cross the BBB, was used as a basis for vascular contamination of brain tissue and was administered to individual groups of rats at a concentration of 10 mg / kg. One hour after injection, animals were sacrificed and plasma and brain were collected and frozen immediately. Following tissue and plasma removal, the concentration of the compound in the brain and plasma was measured using LC-MS / MS. The brain: plasma ratio was calculated as the ratio of concentrations measured in the brain and plasma. The results are shown in FIGS. 16A to 16C.

  Figures 16A, 16B, and 16C show the brain: plasma ratios of various oligomeric mPEGn-O-morphine conjugates, mPEGn-O-codeine conjugates, and PEGn-O-hydroxycodone conjugates, respectively. The brain: plasma ratio of atenolol was shown in each figure and provided the basis for comparison. PEG conjugation resulted in a decrease in the brain: plasma ratio of all of the conjugates compared to the individual unconjugated parent molecule of the conjugate (oxycodone in the case of hydroxycodon). Only PEG-1-morphine showed a greater brain: plasma ratio than its parent, morphine.

(Example 27)
(Time course of brain concentration and plasma concentration of various exemplary mPEG _n -O-opioid conjugates)

  Experiments were performed to determine the concentration of various oligomeric PEG-opioid conjugates in the brain and plasma after IV administration over time.

  The experimental methods and concentrations used were the same as those used for the single time point experiment described in Example 26, but brain and plasma were collected at various different time points.

  All PEG-hydroxycodone conjugates were administered intravenously at 10 mg / kg, while the parent oxycodone was administered intravenously at 1 mg / kg. Brain concentration and plasma concentration and time data for various PEG-opioid conjugates administered are shown in FIGS. 17A-17H (morphine series), FIGS. 18A-18H (codeine series), and FIGS. 19A-19H. Oxycodone / hydroxycodone series).

  The data shows that the maximum increase in brain concentration for all parent molecules and oligomeric PEG-conjugates occurs at the earliest time point, ie 10 minutes after intravenous injection. PEG conjugation results in a significant decrease in brain concentration, and with larger PEG conjugates (≧ PEG-4), brain concentrations remain relatively low over time.

（ｍＰＥＧｎ−ＯＴｓ（ｍＰＥＧｎ−トシラート）の調製（ｎ＝１〜９））

(Example 28)
(Preparation of mPEGn-O-hydrocodonol conjugate)

(Preparation of mPEGn-OTs (mPEGn-tosylate) (n = 1-9))

M-PEGn-OH, which can be dried under high vacuum (even after evaporation of a small amount of heptane or toluene addition), was dissolved in DCM. Toluenesulfonic anhydride (Ts ₂ O, 1.05 eq) and ytterbium (III) triflate (Yb (OTf) ₃ , 0.02 eq) were added and the reaction was allowed to stir overnight (reaction rate was , As fast as 1-5 days to fully consume mPEG _n OH). Once mPEG _n OH was consumed, 2-3 equivalents of polyvinylpyridine was added along with additional DCM to maintain stirring. After more than 24 hours, PVP was filtered off and the filtrate was evaporated, yielding up to 95-100% after full vacuum.

(Preparation of α-6-mPEG _n -O-hydrocodonol conjugate compound (n = 1 to 9))

α-6-mPEG _n —O-hydrocodonol was prepared according to the schematic diagram provided below (where substantially the same approach was used for each of n = 1-9).

(Preparation of hydrocodone free base)

Hydrocodone bitartrate was dissolved in water. To this was added 2 equivalents of solid NaHCO ₃ . Hydrocodone precipitate and dichloromethane were added. The biphasic solution was allowed to stir for 20 minutes. The layers were then separated and the basic aqueous phase was extracted twice with dichloromethane. The organic layer was dried over MgSO ₄ and evaporated to give hydrocodone free base as a white powder. The isolated yield was generally 95 +%.

(Preparation of 6-hydrocodonol free base)

Hydrocodone was dissolved in THF and cooled to -20 ° C. A solution of 1M K-Selectride in THF was added dropwise to the stirring solution over approximately 1 hour. When the reaction was complete, the solution was quenched with 5 equivalents of 1M HCl and the THF was removed in vacuo. The solution was extracted 3 times with ethyl ether. The organic layer was discarded and the acid layer was made alkaline with K ₂ CO ₃ and extracted three times with chloroform. The organic layer was evaporated to give 6-hydrocodonol as a solid. The isolated yield was generally 95 +%. Expected mass = 301.4 M + H = 302.5 Retention time = 0.79 minutes.

(Preparation of 6-hydrocodonol alkalized product with mPEGn-OTs)

Hydrocodonol was dissolved in the minimum amount of anhydrous toluene possible while warming and sonicating. To the room temperature solution, 2 equivalents of NaH (60% dispersion in mineral oil) was added in small portions with good stirring. The mixture was allowed to stir at room temperature for 10 minutes before a solution containing 1.3 equivalents of mPEG _n -OTs in toluene was added over 5 minutes. After 15 minutes at room temperature, the mixture was heated in a 60 ° C. oil bath overnight. LC-MS analysis indicated complete consumption of starting material. The reaction was quenched by pouring the mixture into water and the toluene was removed under vacuum. The aqueous residue was extracted with CHCl ₃ and the aqueous layer was discarded. The combined organic layers were washed with 1/2 saturated NaHCO ₃ and extracted with 1M HCl (aq) with vigorous shaking. The combined aqueous layers were washed with CHCl ₃ and concentrated under vacuum to give the crude product as a dark amber oil.

The residue was purified by reverse phase HPLC using a C8 column. The post-purification yield was generally 25-50% as shown in Table 13.

(Conversion to α-6-mPEG _n- O-hydrocodonol hydrochloride (n = 1-9))

HPLC purified mPEG _n -hydrocodonol TFA salt was dissolved in 1M HCl (aq) and concentrated in vacuo. The residue was redissolved in 1M HCl (aq) and concentrated in vacuo. The residue was azeotroped with acetonitrile three times to give mPEG _n -hydrocodonol HCl salt as a light glass.

The resulting material was purified on a C18 column (Phenomenex Kinetics 50 × 3.0), where the column temperature was 40 ° C., the flow rate was 1.5 mL / min, and mobile phase A was 0.1% TFA / water, mobile phase B is 0.1% TFA / acetonitrile, gradient follows from 5% B to 100% B over 4 minutes, stops at 100% B for 1 minute, then 5% B Equilibrated over 1 minute. The purification results are provided in Table 14.

IC ₅₀ values were determined for each α-6-mPEG _n —O-hydrocodonol compound using a conventional in vitro mu opioid receptor binding affinity assay. The results are provided in Table 15.

Claims (20)

formula:

(Wherein n is an integer having a value of 1 to 9) and pharmaceutically acceptable salts thereof.   The compound according to claim 1, wherein n is 1.   The compound of claim 1, wherein n is 2.   The compound of claim 1, wherein n is 3.   The compound of claim 1, wherein n is 4.   The compound according to claim 1, wherein n is 5.   2. The compound of claim 1, wherein n is 6.   The compound of claim 1, wherein n is 7.   The compound according to claim 1, wherein n is 8.   The compound according to claim 1, wherein n is 9. formula:

(Wherein n is an integer having a value of 1 to 9) and pharmaceutically acceptable salts thereof.   The compound according to claim 11, wherein n is 1.   12. A compound according to claim 11, wherein n is 2.   12. A compound according to claim 11 wherein n is 3.   12. A compound according to claim 11 wherein n is 4.   12. A compound according to claim 11 wherein n is 5.   12. A compound according to claim 11 wherein n is 6.   12. A compound according to claim 11 wherein n is 7.   12. A compound according to claim 11 wherein n is 8.   12. A compound according to claim 11, wherein n is 9.

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